ES2385054T3 - Specific binding proteins for insulin-like growth factors and uses thereof - Google Patents

Abstract

Fully human isolated specific binding protein that preferentially binds to the insulin-II-type decreasing factor (IGF-II) with cross-reactivity for insulin-like growth factor I (IGF-I) and neutralizes the activity of IGF-I and IGF-II, said binding protein, or junction fragment thereof, comprising: a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of "Ser Tyr Asp Ile Asn" (SEQ ID NO: 33); a heavy chain complementarity determining region 2 (CDR2) having the amino acid sequence of "Trp Met Asn Pro Asn Ser Gly Asn Thr Gly Tyr Ala Gln Lys Phe Gln Gly" (SEQ ID NO: 34); a complementarity determining region heavy chain 3 (CDR3) having the amino acid sequence of "Asp Pro Tyr Tyr Tyr Tyr Tyr Gly Met Asp Val" (SEQ ID NO: 35); a light chain complementarity determining region 1 (CDR1) having the sequence deamino acids of "Ser Gly Ser Ser Ser As As n Ile Glu Asn Asn His Val Ser "(SEQ ID NO: 36); a light chain complementarity determining region 2 (CDR2) having the amino acid sequence of" Asp Asn Asn Lys Arg Pro Ser "(SEQ ID NO: 37 ); and a light chain complementarity determining region 3 (CDR3) having the amino acid sequence of "Glu Thr Trp Asp Thr Ser Leu Ser Ala Gly Arg Val" (SEQ ID NO: 38).

Description

Specific binding proteins for insulin-like growth factors and uses thereof.

Cross reference to related requests

This application claims priority according to U.S. 35. §119 regarding the US provisional application with serial number 60 / 750.085, filed on December 13, 2005; US provisional application with serial number 60 / 750,772, filed on December 14, 2005; US provisional application with serial number 60 / 774,747, filed on February 17, 2005; and the US provisional application with serial number 60 / 808,183, filed on May 24, 2006.

Background of the invention

Field of the Invention

The invention relates to binding proteins that bind to the insulin-like growth factor-2 (IGF-II) with cross-reactivity for the insulin-like growth factor-1 (IGF-I) and to uses of such binding proteins . More specifically, the invention relates to monoclonal antibodies directed to IGF-II with cross reactivity by IGF-I and to uses of these antibodies. Aspects of the invention also relate to hybridomas or other cell lines that express such antibodies.

Description of the related technique

The insulin-like growth factor IGF-I and IGF-II are small polypeptides involved in the regulation of cell proliferation, survival, differentiation and transformation. IGFs perform their various actions by interacting primarily with a specific cell surface receptor, the IGF-I receptor (IGF-IR) and activating various intracellular signaling cascades. IGFs circulate in the serum primarily bound to IGF-binding proteins (IGFBP-1 to 6). The interaction of IGF with the IGF-IR is regulated by the IGFBPs, and the IGFs can only bind to the IGF-IR once they are released from the IGFBPs (mainly by proteolysis of the IGFBPs). IGF-I can also bind to a hybrid receptor composed of subunits of IGF-IR and insulin receptor (IR). It has also been shown that IGF-II binds to the "A" isoform of the insulin receptor.

Malignant transformation involves the imbalance of various processes such as cell growth, differentiation, apoptosis and transformation. IGF-I and IGF-II have been implicated in the pathophysiology of a wide range of conditions, and are thought to play a role in tumorigenesis due to the mitogenic and antiapoptotic properties mediated by the IGF-IR receptor: LeRoith and Roberts, Cancer Lett. 195: 127-137 (2003).

IGF-I was discovered as a growth factor produced by the liver under the regulatory control of pituitary growth hormone and was originally called somatomedin (C. Salmon et al., J. Lab. Clin. Med. 49: 825 -826 (1957) Both IGFI and IGF-II express themselves ubiquitously and act as endocrine, paracrine and autocrine growth factors, through their interaction with IGF-IR, a transmembrane tyrosine kinase that is structurally and functionally related with the insulin receptor (IR). IGF-I works primarily by activating IGF-IR, while IGF-II can act through either the IGF-IR or through the IR-isoform.

A. LeRoith and Roberts, Cancer Lett. 195: 127-137 (2003). Additionally, the interaction of both IGF-I and IGF-II with IGF-binding proteins can affect the half-life and availability of IGFs, as well as their direct interaction with receptors in some cases. Rajaram et al., Endocr. Rev. 18: 801-831 (1997).

IGF-I has a long-term impact on cell proliferation, differentiation and apoptosis. Experiments in cultured breast cancer and osteosarcoma cells suggested that IGF-I is a potent mitogen and exerts its mitogenic action by increasing DNA synthesis and stimulating cyclin D1 expression, which accelerates cell cycle progression from phase G1 to S. Furlanetto et al., Mol. Endocrinol 8: 510-517 (1994); Dufourny et al., J. Biol. Chem. 272: 311663-31171 (1997). Suppression of cyclin D1 expression in pancreatic cancer cells abolished the mitogenic effect of IGF-I. Kornmann et al., J. Clin. Invest. 101: 344-352 (1998). In addition to stimulating cell cycle progression, IGF-I also inhibits apoptosis. It was shown that IGF-I stimulated Bcl protein expression and suppressed Bax expression, resulting in an increase in the relative amount of the Bcl / Bax heterodimer, thereby blocking the initiation of the apoptotic pathway. Minshall et al., J. Immunol. 159: 1225-1232 (1997); Parrizas et al., Endocrinology 138: 1355-1358 (1997); Wang et al., Endocrinology 139: 1354-1360 (1998).

Like IGF-I, IGF-II also has mitogenic and antiapoptotic actions and regulates cell proliferation and differentiation. Compared to IGF-I, high concentrations of IGF-II circulate in the serum. High serum IGF-II concentrations have been found in patients with colorectal cancer, with a tendency towards higher concentrations in advanced disease. Renehan et al., Br. J. Cancer 83: 1344-1350. Additionally, the majority of primary tumors and transformed cell lines overexpress mRNA and IGF-II protein. Werner and LeRoith Adv. Cancer Res. 68: 183-223 (1996). Overexpression of IGF-II in colon cancer is associated with an aggressive phenotype, and loss of the footprint (loss of allele-specific expression) of the IGF-II gene may be important in colorectal carcinogenesis. Michell et al., Br. J. Cancer 76: 60-66 (1997); Takano et al., Oncology 59: 210-216 (2000). Cancer cells with a strong tendency to metastasize have four times higher levels of IGF-II expression than cells with a low ability to metastasize. Guerra et al., Int. J. Cancer 65: 812-820 (1996).

Clinical and research studies have highlighted the role of IGF family members in the development, maintenance and progression of cancer. It has been shown that many cancer cells overexpress IGF-IR and / or IGF ligands. For example, IGF-I and IGF-II are strong mitogens for a wide variety of cancer cell lines, including sarcoma, leukemia and cancers of the prostate, breast, lung, colon, stomach, esophagus, liver, pancreas, kidney, thyroid , brain, ovary and uterus. Macaulay et al., Br. J. Cancer 65: 311-320 (1992); Oku et al., Anticancer Res. 11: 1591-1595 (1991); LeRoith et al., Ann. Intern. Med. 122: 54-59 (1995); Yaginuma et al., Oncology 54: 502-507 (1997); Singh et al., Endocrinology 137: 1764-1774 (1996); Frostad et al., Eur. J. Haematol 62: 191-198 (1999). When IGF-I was administered to malignant colon cancer cells, they became resistant to cytokine-induced apoptosis. Remacle-Bonnet et al., Cancer Res. 60: 2007-2017 (2000).

The role of IGFs in cancer is supported by epidemiological studies, which showed that high levels of circulating IGF-I and low levels of IGFBP-3 are associated with an increased risk of developing several common cancers (prostate, breast, colorectal and lung). Mantzoros et al., Br. J. Cancer 76: 1115-1118 (1997); Hankinson et al., Lancet 351: 1393-1396 (1998); Ma et al., J. Natl. Cancer Inst. 91: 620-625 (1999); Karasik et al., J. Clin. Endocrinol Metab. 78: 271-276 (1994). These results suggest that IGF-I and IGF-II act as potent antiapoptotic and mitogenic signals, and that their overexpression correlates with poor prognosis in patients with various types of cancer.

Using deficient mouse models, several studies have also established the role of IGFs in tumor growth. With the development of tissue-specific conditional gene deletion technology, a mouse model of hepatic IGF-I deficiency (LID) was developed. The specific liver deletion of the igf1 gene suppressed the expression of IGF mRNA and caused a drastic reduction in circulating IGF-I levels. Yakar et al., Proc. Natl Acad. Sci. USA 96: 7324-7329 (1999). When mammary tumors were induced in the LID mouse, the reduction in circulating IGF-1 levels resulted in significant reductions in cancer development, growth and metastasis, while the increase in circulating IGF-1 levels was associated with enhanced tumor growth. Wu et al., Cancer Res. 63: 4384-4388 (2003).

Several articles have reported that the inhibition of IGF-IR expression and / or signaling leads to an inhibition of tumor growth, both in vitro and in vivo. It has also been shown that inhibition of IGF signaling increases the susceptibility of tumor cells to chemotherapeutic agents. A variety of strategies (antisense oligonucleotides, soluble receptor, inhibitory peptides, dominant negative receptor mutants, small molecules that inhibit kinase activity and anti-hIGF-IR antibodies) have been developed to inhibit the IGF-IR signaling pathway in cells Tumor One approach has been to target IGF-IR kinase activity with small molecule inhibitors. Recently, two compounds were identified as small molecule kinase inhibitors that could selectively inhibit IGF-IR. Garcia-Echeverria et al., Cancer Cell 5: 231-239 (2004); Mitsiades et al., Cancer Cell 5: 221-230 (2004). Inhibition of IGF-IR kinase activity suppressed the formation of colonies and survival mediated by IGF-I in soft agar of human breast cancer cells MCF-7. Garcia-Echeverria et al., Cancer Cell 5: 231-239 (2004). When an IGF-IR kinase inhibitor was administered to mice carrying tumor xenografts, IGF-IR signaling in tumor xenografts was inhibited and the growth of IGF-IR-driven fibrosarcomas was significantly reduced. Garcia-Echeverria et al., Cancer Cell 5: 231-239 (2004). A similar effect was observed in hematological malignancies, especially multiple myeloma. In multiple myeloma cells, a small molecule IGF-IR kinase inhibitor demonstrated> 16 times greater potency against IGF-1R, compared to the insulin receptor, and was similarly effective in inhibiting growth and cell survival Mitsiades et al., Cancer Cell 5: 221-230 (2004). The same compound was injected intraperitoneally into mice and inhibited the growth of multiple myeloma cells and enhanced the survival of the mice. Mitsiades et al., Cancer Cell 5: 221-230 (2004). When combined with other chemotherapeutic agents at subtherapeutic doses, inhibition of IGF-IR kinase activity synergistically reduced tumor burden. Mitsiades et al., Cancer Cell 5: 221-230 (2004).

Another approach to inhibit IGF signaling has been the development of neutralizing antibodies directed against the IGF-IR receptor. Various groups have developed antibodies against IGF-IR that inhibit IGF-I stimulated autophosphorylation of the receptor, induce internalization and degradation of the receptor and reduce the proliferation and survival of various human cancer cell lines. Hailey et al., Mol Cancer Ther. 1: 1349-1353 (2002); Maloney et al., Cancer Res. 63: 5073-5083 (2003); Benini et al., Clin. Cancer Res. 7: 1790-1797 (2001); Burtrum et al., Cancer Res. 63: 8912-8921 (2003). Additionally, in xenograft tumor models, IGF-IR blockade resulted in significant growth inhibition of breast, kidney and pancreatic tumors in vivo. Bufferrum et al., Cancer Res. 63: 8912-8921 (2003) ; Maloney et al., Cancer Res. 63: 5073-5083 (2003). Experiments using antibodies against humanized IGF-IR produced similar results, inhibiting the growth of breast cancer cells in vitro and in tumor xenografts. Sachdev et al., Cancer Res. 63: 627-635 (2003). Other antibodies against humanized IGF-IR blocked growth inhibition and tyrosine phosphorylation induced by IGF-I in non-small cell and breast lung tumors, as well as in vivo. Cohen et al., Clin. Cancer Res. 11: 2063-2073 (2005); Goetsch et al., Int. J. Cancer 113: 316-328 (2005).

The increase in IGF-I levels has also been associated with several non-cancerous disease states, including acromegaly and gigantism (Barkan, Cleveland Clin. J. Med. 65: 343, 347-349, 1998), while abnormal function of the IGF-I / IGF-II receptor has been implicated in psoriasis (Wraight et al., Nat. Biotech. 18: 521-526, 2000), atherosclerosis and restenosis of the smooth muscle of blood vessels after angioplasty (Bayes-Genis et al., Circ. Res. 86: 125-130, 2000). Increased levels of IGF-I have been implicated in diabetes or complications associated with diabetes, such as microvascular proliferation (Smith et al., Nat. Med. 5: 1390-1395, 1999).

Antibodies against IGF-I and IGF-II have been disclosed in the art. See, for example, Goya et al., Cancer Res. 64: 6252-6258 (2004); Miyamoto et al., Clin. Cancer Res. 11: 3494-3502 (2005). Additionally, see WO 05/18671, WO 05/28515 and WO 03/93317.

Summary

Embodiments of the invention relate to binding proteins that specifically bind to insulin-like growth factors and reduce tumor growth. In one embodiment, the binding proteins are fully human monoclonal antibodies, or binding fragments thereof that specifically bind to insulin-like growth factors and reduce tumor growth. The mechanisms by which this can be achieved may include and are not limited to or inhibition of the binding of IGF-I / II to its IGF-IR receptor, inhibition of IGF-IR signaling induced by IGF-I / II, or either increased clearance of IGF-I / II, thus reducing the effective concentration of IGF-I / II.

Thus, some embodiments provide a completely human isolated specific binding protein according to claim 1 herein. In certain aspects, the binding protein binds to IGF-II with at least 2.5 times more affinity than to IGF-I. In other aspects, the binding protein binds to IGF-II with at least 3, at least 4, at least 5, at least 7, at least 10, at least 50, at least 60, at least 100 or at least 150 times more affinity than IGF-I.

In some embodiments, the specific binding protein has an EC50 of no more than 15 nM to inhibit IGF-I-dependent IGF-I receptor phosphorylation in NIH3T3 cells expressing IGF-1R ectopically. In some aspects, the specific binding protein has an EC50 of no more than 15 nM, no more than 10 nM or no more than 8 nM to inhibit IGF-I-dependent IGF-I receptor phosphorylation in NIH3T3 cells expressing IGF-1R in an ectopic manner.

In some embodiments, the specific binding protein has an EC50 of no more than 5 nM, no more than 4 nM or no more than 3 nM to inhibit IGF-I-dependent IGF-I receptor phosphorylation in NIH3T3 cells expressing IGF-1R in an ectopic manner.

In other embodiments, the specific binding protein inhibits more than 70% of IGF-II-dependent proliferation of NIH3T3 cells expressing recombinant hIGF-IR with an EC50 of no more than 25 nM, no more than 20 nM, no more than 15 nM or not more than 10 nM.

In other embodiments, the specific binding protein inhibits more than 70% of IGF-I-dependent proliferation of NIH3T3 cells expressing recombinant hIGF-IR with an EC50 of no more than 40 nM, no more than 30 nM or no more than 25 nM.

The specific binding protein can be a completely human antibody that binds IGF-I with a Kd of less than 500 picomolar (pM). More preferably, the antibody binds with a Kd of less than 450 picomolar (pM). More preferably, the antibody binds with a Kd less than 410 picomolar (pM). More preferably, the antibody binds with a Kd of less than 350 pM. Even more preferably, the antibody binds with a Kd of less than 300 pM. Affinity and / or greediness measurements can be measured using BIACORE®, as described herein.

The specific binding protein can be a fully human monoclonal antibody that binds IGF-II with a Kd of less than 175 picomolar (pM). More preferably, the antibody binds with a Kd of less than 100 picomolar (pM). More preferably, the antibody binds with a Kd less than 50 picomolar (pM). More preferably, the antibody binds with a Kd of less than 5 picomolar (pM). Even more preferably, the antibody binds with a Kd of less than 2 pM.

In certain embodiments, the specific binding protein is a fully human monoclonal antibody.

or a binding fragment of a fully human monoclonal antibody. Binding fragments may include fragments such as Fab, Fab ’or F (ab’) 2 and Fv.

An embodiment of the invention comprises the fully human monoclonal antibody 7.159.2 (ATCC registration number PTA-7424) that specifically binds IGF-I / II, as discussed in more detail below.

In some embodiments the specific binding protein that binds to insulin-II growth factor (IGF-II) with cross-reactivity for insulin-I growth factor (IGF-I), or binding fragment thereof it can include a heavy chain polypeptide having the sequence of SEQ ID NO .: 6, and a light chain polypeptide having the sequence of SEQ ID NO .: 8.

In certain embodiments, the specific binding protein may be in a mixture with a pharmaceutically acceptable carrier.

Another embodiment includes isolated nucleic acid molecules encoding any of the specific binding proteins described herein, vectors having isolated nucleic acid molecules encoding specific binding proteins, or a host cell transformed with any such such. Nucleic acid vectors and molecules.

In certain embodiments, the specific binding protein that binds to insulin-II growth factor (IGF-II) with cross-reactivity for insulin-I growth factor (IGF-I), or binding fragment of the It does not specifically bind to IGF-II or IGF-I proteins when said proteins are bound to insulin growth factor binding proteins.

Further embodiments include methods for determining the level of insulin-II growth factor (IGF-II) and insulin-I growth factor (IGF-I) in a sample of a patient according to claim 12 herein. document. These methods may include providing a sample of a patient; contacting the sample with a specific binding protein that binds to the insulin-II growth factor (IGF-II) with cross-reactivity for the insulin-I growth factor (IGF-I), or fragment of union thereof; and determine the level of IGFI and IGF-II in said sample. In some aspects, the patient's sample is blood.

Additional embodiments include a conjugate according to claim 18 herein. In some aspects, the therapeutic agent may be a toxin, a radioisotope or a pharmaceutical composition.

Some embodiments provide the use of the specific binding proteins described herein in the preparation of a medicament for the treatment of a malignant tumor. In some aspects, the specific binding protein can be a fully human monoclonal antibody. In certain aspects, the binding protein is AcM 7.159.2 (ATCC registration number PTA-7424). In some aspects, the medicament is for use in combination with a second antineoplastic agent selected from the group consisting of an antibody, a chemotherapeutic agent and a radioactive drug. In some aspects, the drug is for use in conjunction with or after conventional surgery, a bone marrow stem cell transplant or a peripheral stem cell transplant.

The malignant tumor can be melanoma, non-small cell lung cancer, glioma, hepatocellular carcinoma (liver), thyroid tumor, gastric (stomach) cancer, prostate cancer, breast cancer, ovarian cancer, bladder cancer, cancer of lung, glioblastoma, endometrial cancer, kidney cancer, colon cancer, pancreatic cancer and squamous cell carcinoma, for example.

It is indicated that those skilled in the art can easily achieve CDR determinations. See, for example, Kabat et al., Sequences of Proteins of Immunological Interest, fifth edition, publication of NIH 91-3242, Bethesda MD (1991), volumes 1-3.

The embodiments of the invention described herein refer to monoclonal antibodies that bind IGF-I / II and affect the function of IGF-I / II. Other embodiments relate to fully human anti-IGF-I / II antibodies and anti-IGF-I / II antibody preparations with desirable properties from a therapeutic perspective, including high binding affinity for IGF-I / II, the ability to neutralize IGF-I / II in vitro and in vivo and the ability to inhibit cell proliferation induced by IGF-I / II.

Brief description of the drawings

Figure 1 is a graph showing the inhibition of tumor growth of xenograft in nude mice of NIH3T3 cells expressing IGF-II and IGF-IR (clone 32 cells) with the AcM 7.159.2, 7.34.1, 7.251.3 compared to PBS and IgG2 controls. The average tumor volume on the y-axis is shown and the time after implantation on the x-axis is shown.

Figure 2 is a graph showing the body weight in mice with clone xenograft 32 treated with AcM 7.159.2, 7.34.1, 7.251.3 compared to PBS and IgG2 controls. The average body weight on the y-axis is shown and the time after implantation on the x-axis is shown.

Figure 3 is a graph showing the inhibition of tumor growth of xenograft in nude mice of NIH3T3 cells expressing IGFI and IGF-IR (P12 cells) with AcM 7.159.2 compared to PBS control. The mean tumor volume is shown on the y-axis and the time after transplantation (indicated by date) on the x-axis is shown.

Detailed description

The embodiments of the invention described herein refer to binding proteins that specifically bind IGF-II with cross-reactivity by IGF-I (referred to herein as "IGFI / II"). In some embodiments, the binding proteins are antibodies, or binding fragments thereof, and bind to IGF-II with cross-reactivity by IGF-I and inhibit the binding of these proteins to their receptor, IGF-IR. Other embodiments of the invention include completely human neutralizing anti-IGF-I / II antibodies, and antibody preparations that are therapeutically useful and bind to both insulin-like growth factors. Such anti-IGF-I / II antibody preparations preferably have desirable therapeutic properties, including strong binding affinity for IGF-I / II, the ability to neutralize IGF-I / II in vitro and the ability to inhibit IGF-induced cell proliferation. -I / IT in vivo.

Embodiments of the invention also include isolated binding fragments of anti-IGF-I / II antibodies. Preferably, the binding fragments are derived from completely human anti-IGF-I / II antibodies. Exemplary fragments include Fv, Fab ’or other well known antibody fragments, as described in more detail below. Embodiments of the invention also include cells that express fully human antibodies against IGF-I / II. Examples of cells include hybridomas, or recombinantly created cells, such as Chinese hamster ovary (CHO) cells that produce antibodies against IGF-I / II.

Anti-IGF-I / II antibodies are useful for preventing IGF-I / II mediated IGF-I / II signal transduction, thereby inhibiting cell proliferation. The mechanism of action of this inhibition may include the inhibition of the binding of IGF-I / II to its receptor, IGF-IR, the inhibition of IGF-IR signaling induced by IGF-I / II or the enhancement of the clearance of IGF-I / II thus reducing the effective concentration of IGF-I / II for binding to IGF-IR. Diseases that can be treated through this mechanism of inhibition include, but are not limited to, neoplastic diseases, such as melanoma, non-small cell lung cancer, glioma, hepatocellular (liver) carcinoma, gynecological tumors, head cancer and neck, esophageal cancer, glioblastoma and cancers and tumors of the thyroid, stomach, prostate, breast, ovary, bladder, lung, uterus, kidney, colon and pancreas, salivary and colorectal gland.

Other embodiments of the invention include diagnostic tests to specifically determine the amount of IGFI / II in a biological sample. The assay kit may include anti-IGF-I / II antibodies along with the markers necessary to detect such antibodies. These diagnostic tests are useful for examining diseases related to growth factors including, but not limited to, neoplastic diseases, such as melanoma, non-small cell lung cancer, glioma, hepatocellular (liver) carcinoma, gynecological tumors, cancer of head and neck, esophageal cancer, glioblastoma and carcinoma of the thyroid, stomach, prostate, breast, ovary, bladder, lung, uterus, kidney, colon and pancreas, salivary and colorectal gland. Other non-neoplastic pathological conditions may include acromegaly and gigantism, psoariasis, osteoporosis, atherosclerosis and restenosis of the smooth muscle of blood vessels, as well as diabetes.

Embodiments, additional features and the like concerning anti-IGF-I / II antibodies are provided in additional detail below.

Sequence list

Embodiments of the invention include the specific anti-IGF-I / II antibodies listed below in Table 1. This table reports the identification number of each anti-IGF-I / II antibody, together with the SEQ ID number of the corresponding heavy chain and light chain genes. In addition, the germline sequences from which each heavy chain and light chain are derived are also provided in Table 1 below.

Each antibody has been given an identification number that includes either two or three numbers separated by one or two decimal points. In some cases, several clones of an antibody were prepared. Although the clones have the identical amino acid and nucleic acid sequences as the original sequence, they can also be listed separately, with the number of the clone indicated by the number to the right of a second decimal point. Thus, for example, the amino acid and nucleic acid sequences of antibody 7.159.2 are identical to the sequences of antibody 7.159.1.

As can be seen by comparing the sequences in the sequence list, SEQ ID NOs .: 1-20 differ from SEQ ID NOs .: 39-58 because SEQ ID NOs .: 39-58 include regions of untranslated, peptide domains signal and constants for each heavy or light string sequenced.

TABLE 1

AcM ID No .:

Sequence SEQ ID NO:

7,158.1

Nucleotide sequence encoding the heavy chain variable region one

Amino acid sequence that codes for the heavy chain variable region

2

Nucleotide sequence encoding the light chain variable region

3

Amino acid sequence that codes for the light chain variable region

4

7,159.2

Nucleotide sequence encoding the heavy chain variable region 5

Amino acid sequence that codes for the heavy chain variable region

6

Nucleotide sequence encoding the light chain variable region

7

Amino acid sequence that codes for the light chain variable region

8

7.34.1

Nucleotide sequence encoding the heavy chain variable region 9

Amino acid sequence that codes for the heavy chain variable region

10

Nucleotide sequence encoding the light chain variable region

eleven

Amino acid sequence that codes for the light chain variable region

12

7,251.3

Nucleotide sequence encoding the heavy chain variable region 13

Amino acid sequence that codes for the heavy chain variable region

14

Nucleotide sequence encoding the light chain variable region

fifteen

Amino acid sequence that codes for the light chain variable region

16

7,234.1

Nucleotide sequence encoding the heavy chain variable region 17

Amino acid sequence that codes for the heavy chain variable region

18

Nucleotide sequence encoding the light chain variable region

19

Amino acid sequence that codes for the light chain variable region

twenty

7,158.1

Nucleotide sequence encoding the heavy chain variable region 39

Amino acid sequence that codes for the heavy chain variable region

40

Nucleotide sequence encoding the light chain variable region

41

Amino acid sequence that codes for the light chain variable region

42

7,159.2

Nucleotide sequence encoding the heavy chain variable region 43

Amino acid sequence that codes for the heavy chain variable region

44

Nucleotide sequence encoding the light chain variable region

Four. Five

Amino acid sequence that codes for the light chain variable region

46

7.34.1

Nucleotide sequence encoding the heavy chain variable region 47

Amino acid sequence that codes for the heavy chain variable region

48

Nucleotide sequence encoding the light chain variable region

49

Amino acid sequence that codes for the light chain variable region

fifty

7,251.3

Nucleotide sequence encoding the heavy chain variable region 51

Amino acid sequence that codes for the heavy chain variable region

52

Nucleotide sequence encoding the light chain variable region

53

Amino acid sequence that codes for the light chain variable region

54

7,234.1

Nucleotide sequence encoding the heavy chain variable region 55

Amino acid sequence that codes for the heavy chain variable region

56

Nucleotide sequence encoding the light chain variable region

57

Amino acid sequence that codes for the light chain variable region

58

Germ line (7.158.1)

Nucleotide sequence encoding the heavy chain variable region 59

Amino acid sequence that codes for the heavy chain variable region

60

Nucleotide sequence encoding the light chain variable region

61

Amino acid sequence that codes for the light chain variable region

62

Germ line (7.159.1)

Nucleotide sequence encoding the heavy chain variable region 63

Amino acid sequence that codes for the heavy chain variable region

64

Nucleotide sequence encoding the light chain variable region

65

Amino acid sequence that codes for the light chain variable region

66

Germ line (7.34.1)

Nucleotide sequence encoding the heavy chain variable region 67

Amino acid sequence that codes for the heavy chain variable region

68

Nucleotide sequence encoding the light chain variable region

69

Amino acid sequence that codes for the light chain variable region

70

Germ line (7,251.3)

Nucleotide sequence encoding the heavy chain variable region 71

Amino acid sequence that codes for the heavy chain variable region

72

Nucleotide sequence encoding the light chain variable region

73

Amino acid sequence that codes for the light chain variable region

74

Definitions

Unless otherwise defined, the scientific and technical terms used in this document must

5 have the meanings commonly understood by those of ordinary skill in the art. In addition, unless the context requires otherwise, the singular terms must include pluralities and the plural terms must include the singular. In general, the nomenclatures used in connection with, and cell and tissue culture techniques, molecular and chemical biology and hybridization of proteins and oligo or polynucleotides described herein are well known and commonly used in the art.

10 Conventional techniques are used for recombinant DNA, oligonucleotide synthesis and tissue culture and transformation (eg electroporation, lipofection). Enzymatic reactions and purification techniques are performed according to the manufacturer's specifications or as commonly performed in the art or as described herein. The above techniques and procedures are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See for example, Sambrook et al. Molecular Cloning: A Laboratory Manual (3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001)), which is incorporated herein by reference. The nomenclatures used in connection with, and the laboratory procedures and techniques of analytical chemistry, synthetic organic chemistry and medicinal and pharmaceutical chemistry described herein are well known and commonly used in the art. Conventional techniques are used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation and administration, and treatment of patients.

As used according to the present description, the following terms, unless otherwise indicated, should be understood to have the following meanings:

The term "IGF-I" refers to the insulin-like growth factor molecule, and the term "IGF-II" refers to the insulin-II growth factor molecule. The term "IGF-I / II" refers to both molecules, insulin-I and II growth factors, and refers to the preferential binding to IGF-II with cross-reactivity by IGF-I. Thus, an antibody that binds IGF-I / II will preferentially bind IGF-II, but would cross-react with IGF-I, binding to IGF-II with higher affinity than to IGF-I. For example, the antibody can bind IGF-II with 2.5 times more affinity than IGF-I. In certain embodiments, the antibody can bind to IGF-II with at least 5, at least 10, at least 25, at least 50 or at least 150 times more affinity than IGF-I.

The term "neutralizer" when referring to an antibody refers to the ability of an antibody to remove,

or significantly reduce the activity of a target antigen. Accordingly, a neutralizing anti-IGF-I / II antibody can significantly eliminate or reduce the activity of IGF-I / II. A neutralizing antibody against IGF-I / II can act, for example, by blocking the binding of IGF-I / II to its IGF-IR receptor. By blocking this junction, IGF-IR mediated signal transduction is significantly, or completely eliminated. Ideally, a neutralizing antibody against IGF-I / II inhibits cell proliferation.

The term "isolated polynucleotide" as used herein should mean a polynucleotide that has been isolated from its environment in which it occurs naturally. Such polynucleotides can be genomic, cDNA or synthetic. The isolated polynucleotides are preferably not associated with all or a part of the polynucleotides with which they are associated in nature. The isolated polynucleotides can be operatively linked to another polynucleotide with which it is not bound in nature. In addition, isolated polynucleotides preferably do not occur in nature as part of a larger sequence.

The term "isolated protein" referred to herein means a protein that has been isolated from its environment in which it is produced naturally. Such proteins may be derived from genomic DNA, cDNA, recombinant DNA, recombinant RNA or synthetic origin or some combination thereof, that by virtue of their origin, or source of derivation, the "isolated protein" (1) is not associated with proteins found in nature, (2) is free of other proteins from the same source, for example free of murine proteins, (3) is expressed by a cell of a different species or (4) does not occur in nature.

The term "polypeptide" is used herein as a generic term to refer to native protein, fragments or analogs of a polypeptide sequence. Therefore, native protein, fragments and analogs are species of the polypeptide genus. Preferred polypeptides according to the invention comprise human heavy chain immunoglobulin molecules and human kappa light chain immunoglobulin molecules, as well as antibody molecules formed by combinations comprising heavy chain immunoglobulin molecules with light chain immunoglobulin molecules, such as kappa or lambda light chain immunoglobulin molecules, and vice versa, as well as fragments and analogs thereof. Preferred polypeptides according to the invention may also comprise only human heavy chain immunoglobulin molecules or fragments thereof.

The expression "which occurs naturally" as used herein as applied to an object refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and that has not been intentionally modified by man in the laboratory or otherwise occurs naturally .

The term "operably linked" as used herein refers to positions of components thus described that are in a relationship that allows them to function in their intended manner. For example, a control sequence "operably linked" to a coding sequence is connected such that the expression of the coding sequence is achieved under conditions compatible with the control sequences.

The term "polynucleotide" as referred to herein means a polymeric form of nucleotides of at least 10 bases in length, either ribonucleotides or deoxynucleotides or a modified form of any type of nucleotide, or RNA heteroduplex. DNA The term includes single and double stranded forms of DNA.

The term "oligonucleotide" referred to herein includes naturally occurring and modified nucleotides linked to each other by naturally occurring and non-naturally occurring bonds. Oligonucleotides are a subset of polynucleotides that generally comprise a length of 200 bases or less. Preferably, the oligonucleotides are 10 to 60 bases in length and most preferably 12, 13, 14, 15, 16, 17, 18, 19 or 20 to 40 bases in length. Oligonucleotides are usually single stranded, for example for probes; although oligonucleotides can be double stranded, for example for use in the construction of a gene mutant. The oligonucleotides can be oligonucleotides

either felt or antisense.

The term "naturally occurring nucleotides" referred to herein includes deoxyribonucleotides and ribonucleotides. The term "modified nucleotides" referred to herein includes nucleotides with modified or substituted sugar groups and the like. The term "oligonucleotide bonds" referred to herein includes oligonucleotide linkages such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoranilothioate, phosphoranylate, phosphoramidate and the like. See for example, LaPlanche et al. Nucl. Acids Res. 14: 9081 (1986); Stec et al. J. Am. Chem. Soc. 106: 6077 (1984); Stein et al. Nucl. Acids Res. 16: 3209 (1988); Zon et al. Anti-Cancer Drug Design 6: 539 (1991); Zon et al. Oligonucleotides and Analogues: A Practical Approach, p. 87-108 (F. Eckstein, Ed., Oxford University Press, Oxford England (1991)); Stec et al. U.S. Patent No. 5,151,510; Uhlmann and Peyman Chemical Reviews 90: 543 (1990), whose descriptions are incorporated herein by reference. An oligonucleotide may include a marker for detection if desired.

The term "selectively hybridize" referred to herein means binding in a detectable and specific manner. Polynucleotides, oligonucleotides and fragments thereof selectively hybridize with nucleic acid strands under hybridization and washing conditions that minimize appreciable amounts of detectable binding to non-specific nucleic acids. High stringency conditions can be used to achieve selective hybridization conditions as are known in the art and are discussed herein. Generally, the nucleic acid sequence homology between polynucleotides, oligonucleotides or antibody fragments and a nucleic acid sequence of interest will be at least 80%, and more usually with preferably increasing homologies of at least 85%, 90 %, 95%, 99% and 100%.

Two amino acid sequences are "homologous" if there is a partial or complete identity between their sequences. For example, an 85% homology means that 85% of the amino acids are identical when the two sequences are aligned for maximum mating. Gaps are allowed (in any of the two sequences that are mating) in maximum mating; gap lengths of 5 or less are preferred with 2 or less being preferred. Alternatively and preferably, two protein sequences (or polypeptide sequences derived therefrom at least about 30 amino acids in length) are homologous, as this term is used herein, if they have an alignment score of at most 5 (in standard deviation units) using the ALIGN program with the mutation data matrix and a gap penalty of 6 or more. See Dayhoff, M.O., in Atlas of Protein Sequence and Structure, p. 101-110 (Volume 5, National Biomedical Research Foundation (1972)) and Supplement 2 to this volume, p. 1-10. The two sequences or parts thereof are more preferably homologous if their amino acids are more than or equal to 50% identical when aligned optimally using the ALIGN program. It should be noted that there may be regions of homology that differ within two ortholog sequences. For example, the functional sites of mouse and human orthologs may have a higher degree of homology than non-functional regions.

The term "corresponds to" is used herein meaning that a polynucleotide sequence is homologous (ie, it is identical, not strictly evolutionarily related) to all or a part of a reference polynucleotide sequence, or that a sequence of Polypeptide is identical to a reference polypeptide sequence.

In contrast, the term "complementary to" is used herein meaning that the complementary sequence is homologous to all or a part of a reference polynucleotide sequence. For illustration, the "TATAC" nucleotide sequence corresponds to a "TATAC" reference sequence and is complementary to a "GTATA" reference sequence.

The following terms are used to describe the sequence relationships between two or more polynucleotide sequences or amino acids: "reference sequence", "comparison window", "sequence identity", "percentage sequence identity" and "substantial identity " A "reference sequence" is a defined sequence used as the basis for a sequence comparison. A reference sequence may be a subset of a larger sequence, for example, as a segment of a full length gene or cDNA sequence provided in a sequence list or it may comprise a complete gene or cDNA sequence. Generally, a reference sequence is at least 18 nucleotides or 6 amino acids in length, often at least 24 nucleotides or 8 amino acids in length and often at least 48 nucleotides or 16 amino acids in length. Since two polynucleotides or amino acid sequences may each comprise (1) a sequence (i.e., a part of the complete polynucleotide or amino acid sequence) that is similar between the two molecules, and (2) may further comprise a sequence that is divergent between the two polynucleotides or amino acid sequences, sequence comparisons between two (or more) molecules are usually performed by comparing sequences of the two molecules along a "comparison window" to identify and compare local regions of sequence similarity . A "comparison window", as used herein, refers to a conceptual segment of at least about 18 contiguous nucleotide positions or about 6 amino acids in which the polynucleotide sequence or amino acid sequence is compared with a reference sequence of at least 18 contiguous nucleotides or sequences of 6 amino acids and in which the portion of the polynucleotide sequence in the comparison window may include additions, deletions, substitutions and the like (i.e., gaps) of 20 percent or less compared to the reference sequence (which does not include additions or deletions) for optimal alignment of the two sequences. Optimal sequence alignment to align a comparison window can be performed using the local homology algorithm of Smith and Waterman Adv. Appl. Math 2: 482 (1981), using the homology alignment algorithm of Needleman and Wunsch J. Mol. Biol. 48: 443 (1970), by the similarity search method of Pearson and Lipman Proc. Natl Acad. Sci. (USA) 85: 2444 (1988), through computerized implementations of these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package version 7.0, (Genetics Computer Group, 575 Science Dr., Madison, Wis.) , GENEWORKS ™ or MACVECTOR® software packages), or by inspection, and the best alignment is selected (that is, the one that results in the highest percentage of homology throughout the comparison window) generated by the various methods.

The term "sequence identity" means that two polynucleotide sequences or amino acids are identical (that is, on a nucleotide-to-nucleotide or residue-to-residue basis) along the comparison window. The term "sequence identity percentage" is calculated by comparing two optimally aligned sequences throughout the comparison window, determining the number of positions in which the identical amino acid or nucleic acid base residue appears (e.g., A, T, C, G, U or I) in both sequences to provide the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window (that is, the window size) and multiplying the result by 100 to provide the percent sequence identity. The term "substantial identity" as used herein indicates a characteristic of a polynucleotide or amino acid sequence, wherein the polynucleotide or amino acid comprises a sequence that has at least 85 percent sequence identity, preferably at less than 90 to 95 percent sequence identity, more preferably at least 99 percent sequence identity, compared to a reference sequence along a comparison window of at least 18 nucleotide positions (6 of amino acid), often along a window of at least 24-48 nucleotide positions (8-16 amino acid), in which the percentage of sequence identity is calculated by comparing the reference sequence with the sequence that may include deletions or additions that amount to 20 percent or less of the reference sequence along the comparison window. The reference sequence may be a subset of a larger sequence.

As used herein, the twenty conventional amino acids and their abbreviations follow conventional use, see Immunology-A Synthesis (2nd edition, ES Golub and DR Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)) , which is incorporated herein by reference. Stereoisomers (for example, D-amino acids) of the twenty conventional amino acids, unnatural amino acids such as a-, a-disubstituted amino acids, N-alkylamino acids, lactic acid and other unconventional amino acids may also be suitable components for polypeptides of the present invention . Examples of unconventional amino acids include: 4-hydroxyproline, y-carboxyglutamate, sN, N, N-trimethyl-lysine, sN-acetyl-lysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5- hydroxylysine, cN-methylarginine and other similar amino acids and imino acids (for example, 4-hydroxyproline). In the polypeptide notation used herein, the left hand address is the amino terminal address and the right hand address is the carboxyl terminal address, according to the convention and conventional use.

Similarly, unless otherwise specified, the left-hand end of single stranded polynucleotide sequences is the 5 ′ end; The left-hand direction of double-stranded polynucleotide sequences is called the 5 ’direction. The 5 ’to 3’ address of nascent RNA transcripts is called the transcript address; Sequence regions in the DNA strand that have the same sequence as the RNA and that are 5 'from the 5' end of the RNA transcript are called "sequences in the sense of 5 '", sequence regions in the strand of DNA that have the same sequence as the RNA and that are 3 'from the 3' end of the RNA transcript are called "sequences in the sense of 3 '".

As applied to polypeptides, the term "substantial identity" means that two peptide sequences, when optimally aligned, such as by GAP or BESTFIT programs using default gap weights, share a sequence identity of at least 80 percent, preferably a sequence identity of at least 90 percent, more preferably a sequence identity of at least 95 percent and most preferably a sequence identity of at least 99 percent. Preferably, residue positions that are not identical differ by conservative amino acid substitutions.

Conservative amino acid substitutions refer to the ability to exchange residues that have similar side chains. For example, a group of amino acids that have aliphatic side chains is glycine, alanine, valine, leucine and isoleucine; a group of amino acids that have aliphatic hydroxyl side chains is serine and threonine; A group of amino acids that have amide-containing side chains is asparagine and glutamine; a group of amino acids that have aromatic side chains is phenylalanine, tyrosine and tryptophan; a group of amino acids that have basic side chains is lysine, arginine and histidine; and a group of amino acids that have sulfur-containing side chains is cysteine and methionine. Preferred conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic-aspartic and asparagine-glutamine.

As discussed herein, it is contemplated that minor variations in the amino acid sequences of antibodies or immunoglobulin molecules are encompassed by the present invention, provided that such variations in the amino acid sequence maintain a sequence identity of at least 75%, more preferably at least 80%, 90%, 95% and most preferably 99% with the antibodies or immunoglobulin molecules described herein. In particular, conservative amino acid substitutions are contemplated. Conservative substitutions are those that take place within a family of amino acids that have related side chains. Genetically encoded amino acids are generally divided into families: (1) acid = aspartate, glutamate; (2) basic = lysine, arginine, histidine; (3) non-polar = alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar = glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Most preferred families are: serine and threonine are an aliphatic hydroxyl family; asparagine and glutamine are a family that contains amide; Alanine, valine, leucine and isoleucine are an aliphatic family; and phenylalanine, tryptophan and tyrosine are an aromatic family. For example, it is reasonable to expect that an isolated substitution of a leucine for an isoleucine or valine, an aspartate for a glutamate, a threonine for a serine or a similar substitution of an amino acid for a structurally related amino acid will not have an important effect on function. binding or properties of the resulting molecule, especially if the substitution does not imply an amino acid within a framework site. It can be determined if an amino acid change results in a functional peptide by testing the specific activity of the polypeptide derivative. Essays are described in detail herein. Those of ordinary skill in the art can easily prepare fragments or analogs of antibodies or immunoglobulin molecules. Preferred amino and carboxyl termini of fragments or analogs occur near the limits of functional domains. Structural and functional domains can be identified by comparing nucleotide and / or amino acid sequence data with public or registered sequence databases. Preferably, computerized comparison methods are used to identify sequence motifs or conformation domains of predicted proteins that are produced in other proteins of known structure and / or function. Methods for identifying protein sequences that fold to give a known three-dimensional structure are known. Bowie et al. Science 253: 164 (1991). Therefore, the above examples demonstrate that those skilled in the art can recognize sequence motifs and structural conformations that can be used to define structural and functional domains according to the antibodies described herein.

Conservative amino acid substitutions are those that: (1) reduce the susceptibility to proteolysis, (2) reduce the susceptibility to oxidation, (3) alter the binding affinity to form protein complexes, (4) alter the binding affinities and (5) confer or modify other physicochemical or functional properties of such analogs. The analogs may include various muteins of a sequence other than the naturally occurring peptide sequence. For example, individual or multiple amino acid substitutions (preferably conservative amino acid substitutions) can be made in the sequence that occurs naturally (preferably in the part of the polypeptide outside the domain (s) that forms intermolecular contacts (n) A conservative amino acid substitution should not substantially change the structural characteristics of the original sequence (for example, a substitution amino acid should not tend to break a helix that appears in the original sequence, nor alter other types of secondary structure that characterizes the original sequence.) Examples of secondary and tertiary polypeptide structures recognized in the art are described in Proteins, Structures and Molecular Principles (Creighton, Ed., WH Freeman and Company, New York (1984)); Introduction to Protein Structure (C. Branden and J. Tooze, eds., Garland Publishing, New York, NY (1991)); and Thornton et al. Nature 354: 105 (1991), which are incorporated herein by reference.

The term "polypeptide fragment" as used herein refers to a polypeptide having an amino terminal and / or carboxyl terminal deletion, but in which the remaining amino acid sequence is identical to the corresponding positions in the sequence. which occurs naturally deduced, for example, from a full length cDNA sequence. The fragments are at least 5, 6, 8 or 10 amino acids in length, preferably at least 14 amino acids in length, more preferably at least 20 amino acids in length, usually at least 50 amino acids in length and even more preferably at least 70 amino acids in length . The term "analog" as used herein refers to polypeptides that are composed of a segment of at least 25 amino acids that has substantial identity with a portion of a deduced amino acid sequence and that has at least one of the following properties: (1) specific binding to IGF-I / II, under suitable binding conditions, (2) ability to block the binding to appropriate IGF-I / II or

(3) ability to inhibit IGF-I / II activity. Typically, polypeptide analogs comprise a conservative amino acid substitution (or addition or deletion) with respect to the sequence that occurs naturally. The analogs are usually at least 20 amino acids in length, preferably at least 50 amino acids in length or more, and can often be as long as a naturally occurring full length polypeptide.

Peptide analogs are commonly used in the pharmaceutical industry as non-peptide drugs with properties analogous to those of the template peptide. These types of non-peptide compound are called "peptide mimetics" or "peptidomimetics." Fauchere, J. Adv. Drug Res. 15:29 (1986); Veber and Freidinger TINS p.392 (1985); and Evans et al. J. Med. Chem. 30: 1229 (1987), which are incorporated herein by reference. Such compounds are often developed with the help of computerized molecular modeling. Peptide mimetics that are structurally similar to therapeutically useful peptides can be used to produce a therapeutic affect

or equivalent prophylactic. Generally, peptidomimetics are structurally similar to a model polypeptide (i.e., a polypeptide that has a biochemical property or pharmacological activity), such as a human antibody, but that has one or more peptide bonds optionally substituted by a bond selected from the group consisting of in: --CH2NH--, --CH2S--, --CH2-CH2--, --CH = CH - (cis and trans), - COCH2--, --CH (OH) CH2 - and -CH2SO--, by methods well known in the art. Systematic substitution of one or more amino acids of a consensus sequence can be used with a D-amino acid of the same type (eg, D-lysine instead of Llisin) to generate more stable peptides. In addition, restricted peptides comprising a consensus sequence or a substantially identical consensus sequence variation can be generated by methods known in the art (Rizo and Gierasch Ann. Rev. Biochem. 61: 387 (1992), incorporated herein by reference) ; for example, by adding internal cysteine residues that can form intramolecular disulfide bridges that cycle the peptide.

As used herein, the term "antibody" refers to a polypeptide or group of polypeptides that are composed of at least one binding domain that is formed from the folding of polypeptide chains that have three-dimensional binding spaces with internal surface forms and load distributions complementary to the characteristics of an antigenic determinant of an antigen. An antibody normally has a tetrameric form, comprising two identical pairs of polypeptide chains, each pair having a "light" and a "heavy" chain. The variable regions of each light / heavy chain pair form an antibody binding site.

"Binding fragments" of an antibody are produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies. Binding fragments include Fab, Fab ’, F (ab’) 2, Fv and single chain antibodies. It is understood that a "bispecific" or "bifunctional" antibody has each of its identical binding sites. An antibody substantially inhibits the adhesion of a receptor to a counter receptor when an excess of antibody reduces the amount of receptor bound to the counter receptor by at least about 20%, 40%, 60% or 80%, and more usually more than approximately 85% (as measured in an in vitro competitive binding assay).

As used herein, a "binding protein" or a "specific binding protein" are proteins that specifically bind to a target molecule. Antibodies, and antibody binding fragments, are binding proteins.

The term "epitope" includes any protein determinant that can specifically bind to an immunoglobulin or T-cell receptor. Epitopic determinants usually consist of groups of chemically active surface molecules such as amino acids or sugar side chains and may have, but not be always, specific three-dimensional structural characteristics, as well as specific load characteristics. It is said that an antibody specifically binds to an antigen when the dissociation constant is: 1 mM, preferably: 100 nM and most preferably: 10 nM.

The term "agent" is used herein to indicate a chemical compound, a mixture of chemical compounds, a biological macromolecule or an extract prepared from biological materials.

"Active" or "activity" with respect to an IGF-I / II polypeptide refers to a part of an IGF-I / II polypeptide that has a biological activity or an immunological activity of an IGF-I / II polypeptide. native. "Biological" when used herein refers to a biological function that results from the activity of the native IGF-I / II polypeptide. A preferred biological activity of IGF-I / II includes, for example, cell proliferation induced by IGF-I / II.

"Mammalian" when used herein refers to any animal that is considered a mammal. Preferably, the mammal is a human being.

Digestion of antibodies with the papain enzyme results in two identical antigen binding fragments, also known as "Fab" fragments, and an "Fc" fragment, which has no antigen binding activity but has the ability to crystallize. Digestion of antibodies with the enzyme pepsin results in an F (ab ’) 2 fragment in which the two arms of the antibody molecule remain attached and comprise two antigen binding sites. F (ab ’) 2 fragment has the ability to cross-link antigen.

"Fv", when used herein, refers to the minimum fragment of an antibody that retains both antigen recognition and antigen binding sites.

"Fab", when used herein, refers to a fragment of an antibody comprising the constant domain of the light chain and the CH1 domain of the heavy chain.

The term "AcM" refers to a monoclonal antibody.

"Liposome," when used herein, refers to a small vesicle that may be useful for the administration of drugs that may include the IGF-I / II polypeptide of the invention or antibodies against an IGF-polypeptide. I / II of this type to a mammal.

"Marker" or "labeled", as used herein, refers to the addition of a detectable moiety to a polypeptide, for example, a radiolabel, fluorescent label, chemiluminescently labeled enzyme marker or a biotinyl group. Radioisotopes or radionuclides may include 3H, 14C, 15N, 35S, 90Y, 99Tc,

125I,

111In, 131I, fluorescent markers may include rhodamine, lanthanide phosphors or FITC and enzymatic markers may include horseradish peroxidase, 1-galactosidase, luciferase, alkaline phosphatase.

The term "drug or pharmaceutical agent" as used herein refers to a composition.

or chemical compound that can induce a desired therapeutic effect when properly administered to a patient. Other terms of chemistry are used herein according to conventional use in the art, as shown by way of example by The McGraw-Hill Dictionary of Chemical Terms (Parker, S., Ed., McGraw-Hill, San Francisco (1985)) (incorporated herein by reference).

As used herein, "substantially pure" means that an object species is the predominant species present (ie, on a molar basis it is more abundant than any other individual species in the composition), and preferably a substantially purified fraction. It is a composition in which the target species comprises at least about 50 percent (on a molar basis) of all macromolecular species present. Generally, a substantially pure composition will comprise more than about 80 percent of all macromolecular species present in the composition, more preferably more than about 85%, 90%, 95% and 99%. Most preferably, the target species is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) in which the composition consists essentially of a single macromolecular species.

The term "patient" includes human and veterinary subjects.

Human antibodies and antibody humanization

Human antibodies avoid some of the problems associated with antibodies that have variable and / or constant rat or murine regions. The presence of such murine or rat-derived proteins can lead to rapid clearance of antibodies or can lead to the generation of an immune response against the antibody by a patient. In order to avoid the use of murine antibodies or rat derivatives, completely human antibodies can be generated through the introduction of a locus of functional human antibodies in a rodent, another mammal or animal so that the rodent, another mammal or animal produces completely human antibodies.

One method for generating fully human antibodies is through the use of XenoMouse® strains of mice that have been engineered to contain configured fragments of the germ line with a size of up to but less than 1000 kb from the human heavy chain locus. and the human light chain locus. See Mendez et al. Nature Genetics 15: 146-156 (1997) and Green and Jakobovits J. Exp. Med. 188: 483495 (1998). XenoMouse® strains are available from Abgenix, Inc. (Fremont, CA).

The production of XenoMouse® strains of mice is discussed and further outlined in US patent applications with serial numbers 07 / 466.008, filed January 12, 1990; 07 / 610,515, filed on November 8, 1990, 07 / 919,297, filed on July 24, 1992, 07 / 922,649, filed on July 30, 1992, 08 / 031,801, filed on March 15, 1993, 08 / 112,848, filed on August 27, 1993, 08 / 234,145, filed on April 28, 1994, 08 / 376,279, filed on January 20, 1995, 08 / 430,938, filed on April 27, 1995, 08 / 464,584, filed June 5, 1995, 08 / 464,582, filed June 5, 1995, 08 / 463,191, filed June 5, 1995, 08 / 462,837, filed June 5, 1995, 08 / 486,853, filed on June 5, 1995, 08 / 486,857, filed on June 5, 1995, 08 / 486,859, filed on June 5, 1995, 08 / 462,513, filed on June 5, 1995, 08 / 724,752, filed on 2 October 1996, 08 / 759,620, filed on December 3, 1996, U.S. publication 2003/0093820, filed on November 30, 2001, and U.S. Patent Nos. 6,162,963, 6,150,584, 6,114,598, 6 . 075,181 and

5,939,598 and Japanese patents No. 3 068 180 B2, 3 068 506 B2 and 3 068 507 B2. See also European Patent No. EP 0 463 151 B1, granted and published on June 12, 1996, International Patent Application No.

WO 94/02602, published on February 3, 1994, International Patent Application No. WO96 / 34096, published on October 31, 1996, WO98 / 24893, published on June 11, 1998, WO00 / 76310, published on December 21, 2000. The descriptions of each of the patents, applications and references cited above are incorporated herein by reference in their entirety.

In an alternative approach, others, including GenPharm International, Inc., have used a "minilocus" approach. In this minilocus approach, an exogenous Ig locus is mimicked through the inclusion of chunks (individual genes) of the Ig locus. Thus, one or more VH genes, one or more DH genes, one or more JH genes, a constant mu region and usually a second constant region (preferably a gamma constant region) are formed to give a construct for insertion. In an animal. This approach is described in U.S. Patent No.

5,545,807 granted to Surani et al. and U.S. Patent Nos. 5,545,806, 5,625,825, 5,625,126, 5,633,425, 5,661,016, 5,770,429, 5,789,650, 5,814,318, 5,877,397, 5,874,299 and 6,255,458 each granted to Lonberg and Kay, U.S. Patent No. 5,591,669 and 6,023,010 issued to Krimpenfort and Berns, U.S. Patent Nos. 5,612,205, 5,721,367 and 5,789,215 issued to Berns et al. , and U.S. Patent No.

5,643,763 granted to Choi and Dunn, and GenPharm international US patent applications with serial numbers 07 / 574,748, filed August 29, 1990, 07 / 575,962, filed August 31, 1990, 07 / 810,279, filed on December 17, 1991, 07 / 853.408, filed on March 18, 1992, 07 / 904.068, filed on June 23, 1992, 07 / 990.860, filed on December 16, 1992, 08 / 053.131, filed on April 26, 1993, 08 / 096,762, filed on July 22, 1993, 08 / 155,301, filed on November 18, 1993, 08 / 161,739, filed on December 3, 1993, 08 / 165,699, filed on 10 December 1993, 08 / 209,741, filed on March 9, 1994, whose descriptions are incorporated herein by reference. See also European Patent No. 0 546 073 B1, International Patent Applications No. WO 92/03918, WO 92/22645, WO 92/22647, WO 92/22670, WO 93/12227, WO 94/00569 , WO 94/25585, WO 96/14436, WO 97/13852 and WO 98/24884 and U.S. Patent No. 5,981,175, the description of which is incorporated herein by reference in its entirety. See also Taylor et al., 1992, Chen et al., 1993, Tuaillon et al., 1993, Choi et al., 1993, Lonberg et al., (1994), Taylor et al., (1994) and Tuaillon et al., (1995), Fishwild et al., (1996), whose descriptions are incorporated herein by reference in their entirety.

Kirin has also demonstrated the generation of human antibodies from mice in which, through microcellular fusion, large pieces of chromosomes, or complete chromosomes, have been introduced. See European Patent Applications No. 773 288 and 843 961, whose descriptions are incorporated herein by reference. Additionally, KMT mice have been generated, which are the result of the crossing of Kirin Tc mice with mice with Medarex minilocus (Humab). These mice have the human IgH transchromosome of Kirin mice and the kappa chain transgene of Genpharm mice (Ishida et al., Cloning Stem Cells, (2002) 4: 91-102).

Human antibodies can also be derived by in vitro methods. Suitable examples include but are not limited to phage display (CAT, Morphosys, Dyax, Biosite / Medarex, Xoma, Symphogen, Alexion (formerly Proliferon), Affimed), ribosome presentation (CAT), yeast presentation and the like.

Antibody preparation

Antibodies were prepared, as described herein, through the use of XenoMouse® technology, as described below. Such mice, then, can produce antibodies and human immunoglobulin molecules and are deficient in the production of murine immunoglobulin antibodies and molecules. The technologies used to achieve this are disclosed in the patents, applications and references disclosed in the background section of this document. In particular, however, a preferred embodiment of transgenic production of mice and antibodies therefrom is disclosed in U.S. Patent Application Serial Number 08 / 759,620, filed December 3, 1996 and applications for International Patents Nos. WO 98/24893, published June 11, 1998 and WO 00/76310, published December 21, 2000, the descriptions of which are incorporated herein by reference. See also Mendez et al. Nature Genetics 15: 146-156 (1997), the description of which is incorporated herein by reference.

Through the use of such technology, fully human monoclonal antibodies against a variety of antigens have been produced. Essentially, XenoMouse® lines of mice are immunized with an antigen of interest (eg IGF-I / II), lymphatic cells (such as B cells) are recovered from the hyperimmunized mice and the recovered lymphocytes are fused with a type cell line myeloid to prepare immortal hybridoma cell lines. These hybridoma cell lines are examined and selected to identify hybridoma cell lines that produce specific antibodies against the antigen of interest. Methods for the production of multiple hybridoma cell lines that produce specific antibodies against IGF-I / II are provided herein. In addition, the characterization of the antibodies produced by such cell lines, including analysis of the amino acid and nucleotide sequence of the heavy and light chains of such antibodies, is provided herein.

Alternatively, instead of fusing with myeloma cells to generate hybridomas, B cells can be tested directly. For example, CD19 + B cells can be isolated from hyperimmune XenoMouse® mice and allowed to proliferate and differentiate to give plasma cells that secrete antibodies. The antibodies are then examined from cell supernatants by ELISA to detect reactivity against the IGF-I / II immunogen. Supernatants could also be examined for immunoreactivity against IGF-I / II fragments to further map the different antibodies to detect binding to domains of functional interest in IGF-I / II. Antibodies can also be screened against other related human chemokines and against the rat, mouse and non-human IGF-I / II orthologs, such as cynomolgus monkey, the latter to determine cross-reactivity of species. B cells from wells containing antibodies of interest can be immortalized by various methods including fusion to prepare hybridomas either from individual or pooled wells, or by infection with EBV or transfection by known immortalization genes and then plating on suitable medium. . Alternatively, individual plasma cells that secrete antibodies with the desired specificities are then isolated using a specific hemolytic plate assay of IGF-I / II (Babcook et al., Proc. Natl. Acad. Sci. USA 93: 7843-48 (1996 )). The cells selected as the target for lysis are preferably sheep red blood cells (SRBC) coated with the IGF-I / II antigen.

In the presence of a B-cell culture containing plasma cells that secrete the immunoglobulin of interest and complement, the formation of a plaque indicates lysis mediated by IGF-I / II specific to the sheep red blood cells surrounding the plasma cell of interest . The individual antigen specific plasma cell in the center of the plate can be isolated and the genetic information encoding the antibody specificity is isolated from the individual plasma cell. Using reverse transcription followed by PCR (RT-PCR), the DNA encoding the variable heavy and light chain regions of the antibody can be cloned. Such cloned DNA can then be further inserted into a suitable expression vector, preferably a vector cassette such as a cDNA, more preferably a cDNA vector of this type containing the heavy and light chain constant immunoglobulin domains. The generated vector can then be transfected into host cells, for example HEK293 cells, CHO cells, and cultured in conventional nutrient media modified as appropriate to induce transcription, select transformants or amplify the genes encoding the desired sequences.

In general, the antibodies produced by the fused hybridomas were heavy chains of human IgG2 with fully human kappa or lambda light chains. The antibodies described herein have heavy chains of human IgG4 as well as heavy chains of IgG2. The antibodies can also be from other human isotypes, including IgG1. The antibodies had high affinities, normally having a Kd of from about 10-6 to about 10-12 M or less, when measured by dissolution phase and solid phase techniques. Antibodies having a KD of at least 10-11 M are preferred to inhibit IGF-I / II activity.

As will be appreciated, anti-IGF-I / II antibodies can be expressed in cell lines other than hybridoma cell lines. Sequences encoding particular antibodies can be used to transform a suitable mammalian host cell. The transformation can be by any known method of introducing polynucleotides into a host cell, including, for example, packaging the polynucleotide into a virus (or a viral vector) and transducing a host cell with the virus (or vector) or by transfection procedures known in the art, as shown by way of example by U.S. Patent Nos. 4,399,216, 4,912,040, 4,740,461 and 4,959,455 (patents incorporated herein by the present document for reference). The transformation procedure used depends on the host to be transformed. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide (s) in liposomes. and direct microinjection of DNA in nuclei.

Mammalian cell lines available as hosts for expression are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC), including but not limited to Chinese hamster ovary (CHO) cells, cells HeLa, hamster breeding kidney cells (BHK), monkey kidney cells (COS), human hepatocellular carcinoma cells (eg, Hep G2), 293 human epithelial kidney cells and several other cell lines. Cell lines of particular preference are selected through the determination of which cell lines have high levels of expression and produce antibodies with constitutive IGF-I / II binding properties.

Anti-IGF-I / II antibodies are useful in the detection of IGF-I / II in patient samples and are therefore useful as diagnoses for pathological conditions as described herein. In addition, based on their ability to significantly neutralize IGF-I / II activity (as demonstrated in the examples below), anti-IGF-I / II antibodies have therapeutic effects in the treatment of symptoms and conditions that result of the expression of IGF-I / II. In specific embodiments, the antibodies and methods herein refer to the treatment of symptoms that result from cell proliferation induced by IGF-I / II. Additional embodiments involve the use of the antibodies and methods described herein to treat diseases including neoplastic diseases, such as melanoma, non-small cell lung cancer, glioma, hepatocellular carcinoma (liver), thyroid tumor, gastric cancer (stomach). ), prostate cancer, breast cancer, ovarian cancer, bladder cancer, lung cancer, glioblastoma, endometrial cancer, kidney cancer, colon cancer, gynecological tumors, head and neck cancer, esophageal cancer and pancreatic cancer . Other non-neoplastic pathological conditions may include acromegaly and gigantism, psoriasis, osteoporosis, atherosclerosis and restenosis of the smooth muscle of blood vessels, as well as diabetes.

Therapeutic administration and formulations

Embodiments of the invention include sterile pharmaceutical formulations of anti-IGF-I / II antibodies that are useful as treatments for diseases. Such formulations would inhibit the binding of IGF-I / II to its IGF-IR receptor, thereby effectively treating pathological conditions in which, for example, tissue or serum IGF-I / II is abnormally elevated. The anti-IGF-I / II antibodies preferably have an adequate affinity to potently neutralize IGF-I / II, and preferably have an adequate duration of action to allow infrequent dosing in humans. A prolonged duration will also allow less frequent and more convenient dosing schedules by alternative parenteral routes such as subcutaneous or intramuscular injection.

Sterile formulations can be created, for example, by filtration through sterile filtration membranes, before or after lyophilization and reconstitution of the antibody. The antibody will usually be stored in lyophilized form or in solution. Therapeutic antibody compositions are generally placed in a container that has a sterile access point, for example, an intravenous solution vial or bag that has an adapter that allows recovery of the formulation, such as a plug pierceable by a needle. hypodermic injection

The route of administration of the antibody is according to known methods, for example, injection or infusion by intravenous, intraperitoneal, intracerebral, intramuscular, intraocular, intraarterial, intrathecal, inhalation or intralesional routes,

or by sustained release systems as indicated below. The antibody is preferably administered by infusion or by bolus injection.

An effective amount of antibody to be used therapeutically will depend, for example, on the therapeutic objectives, the route of administration and the condition of the patient. Therefore, it is preferred that the therapist titer the dosage and modify the route of administration as required to obtain the optimal therapeutic effect. Normally, the doctor will administer antibody until a dosage is reached that achieves the desired effect. The progress of this therapy is easily monitored by conventional assays or by the assays described herein.

Antibodies, as described herein, can be prepared in a mixture with a pharmaceutically acceptable carrier. This therapeutic composition can be administered intravenously or through the nose or lung, preferably as a powder or liquid aerosol (lyophilized). The composition can also be administered parenterally or subcutaneously as desired. When administered systemically, the therapeutic composition must be sterile, pyrogen-free and must be in a parenterally acceptable solution that it has due to pH, isotonicity and stability. These conditions are known to those skilled in the art. In summary, dosage formulations of the compounds described herein are prepared for storage or administration by mixing the compound having the desired degree of purity with physiologically acceptable carriers, excipients or stabilizers. Such materials are not toxic to the receptors at the dosages and concentrations employed, and include buffers such as TRIS HCl, phosphate, citrate, acetate and other salts of organic acids; antioxidants such as ascorbic acid; low molecular weight peptides (less than about ten residues) such as polyarginine, proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidinone; amino acids such as glycine, glutamic acid, aspartic acid or arginine; monosaccharides, disaccharides and other carbohydrates including cellulose or its derivatives, glucose, mannose or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; counterions such as sodium and / or non-ionic surfactants such as TWEEN, PLURONICS or polyethylene glycol.

Sterile compositions for injection can be formulated according to conventional pharmaceutical practice as described in Remington: The Science and Practice of Pharmacy (20th ed., Lippincott Williams & Wilkens Publishers (2003)). For example, dissolution or suspension of the active compound in a vehicle such as water or vegetable oil that occurs naturally as sesame, peanut or cottonseed oil or a synthetic fatty vehicle such as ethyl oleate or the like may be desired. . Buffers, preservatives, antioxidants and the like may be incorporated according to accepted pharmaceutical practice.

Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing the polypeptide, matrices that are in the form of shaped articles, films or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly (2-hydroxyethyl methacrylate) as described by Langer et al., J. Biomed Mater. Res., (1981) 15: 167-277 and Langer, Chem. Tech., (1982) 12: 98-105, or polyvinyl alcohol, polylactides (U.S. Patent No. 3,773,919,

EP 58,481), copolymers of L-glutamic acid and gamma-ethyl L-glutamate (Sidman et al., Biopolymers, (1983) 22: 547-556); non-degradable ethylene-vinyl acetate copolymers (Langer et al., cited above), degradable lactic acid-glycolic acid such as LUPRON DepotT (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate) and poly ( D - (-) - 3-hydroxybutyric acid (EP 133,988).

While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid allow the release of molecules for more than 100 days, certain hydrogels release proteins for shorter periods of time. When encapsulated proteins remain in the body for a long time, they can be denatured or added as a result of exposure to moisture at 37 ° C, resulting in a loss of biological activity and possible changes in immunogenicity. Rational strategies for protein stabilization can be devised depending on the mechanism involved. For example, if it is discovered that the aggregation mechanism is the formation of intermolecular SS bonds through disulfide exchange, stabilization can be achieved by modifying sulfhydryl residues, lyophilizing from acid solutions, controlling the moisture content, using appropriate additives and developing specific polymer matrix compositions.

Sustained release compositions also include preparations of antibody crystals suspended in suitable formulations that can keep the crystals in suspension. These preparations, when injected subcutaneously or intraperitoneally, can produce a sustained release effect. Other compositions also include antibodies trapped in liposomes. Liposomes containing such antibodies are prepared by methods known per se: U.S. Patent No. 3,218,121; Epstein et al., Proc. Natl Acad. Sci. USA, (1985) 82: 3688-3692; Hwang et al., Proc. Natl Acad. Sci. USA, (1980) 77: 4030-4034; EP 52,322; EP 36,676; EP 88,046; EP 143,949; 142,641; Japanese patent application 83-118008; U.S. Patent Nos. 4,485,045 and 4,544,545; and EP 102,324.

The dosage of the antibody formulations for a given patient will be determined by the attending physician taking into account various factors that are known to modify the action of the drugs including the severity and type of disease, body weight, sex, diet. , the timing and route of administration, other medications and other relevant clinical factors. Therapeutically effective dosages can be determined by methods either in vivo or in vitro.

An effective amount of the antibodies, described herein, to be used therapeutically will depend, for example, on the therapeutic objectives, the route of administration and the condition of the patient. Therefore, it is preferred that the therapist titer the dosage and modify the route of administration as required to obtain the optimal therapeutic effect. A typical daily dosage could range between about 0.001 mg / kg and 100 mg / kg or more, depending on the factors mentioned above. Normally, the doctor will administer the therapeutic antibody until a dosage is reached that achieves the desired effect. The progress of this therapy is easily monitored by conventional assays or as described herein.

It will be appreciated that the administration that administration of therapeutic entities according to the compositions and methods herein will be administered with carriers, excipients and other suitable agents that are incorporated into formulations to provide improved transfer, administration, tolerance and the like. These formulations include, for example, paste powders, ointments, jellies, waxes, oils, lipids, vesicles containing lipids (cationic or anionic) (such as LipofectinT), DNA conjugates, anhydrous absorption pastes, oil-in-water emulsions and of water in oil, carbowax emulsions (polyethylene glycols of various molecular weights), semi-solid gels and semi-solid mixtures containing carbowax. Any of the above mixtures may be appropriate in treatments and therapies according to the present invention, provided that the active ingredient in the formulation is not inactivated by the formulation and the formulation is physiologically compatible and tolerable with the route of administration. See also Baldrick P. “Pharmaceutical excipient development: the need for preclinical guidance”. Regul. Toxicol Pharmacol 32 (2): 210-8 (2000), Wang W. "Lyophilization and development of solid protein pharmaceuticals". Int. J Pharm. 203 (1-2): 1-60 (2000), Charman WN "Lipids, lipophilic drugs, and oral drug delivery-some emerging concepts." J Pharm Sci. 89 (8): 967-78 (2000), Powell et to the. “Compendium of excipients for parenteral formulations” PDA J Pharm Sci Technol. 52: 238-311 (1998) and the references therein for additional information related to formulations, excipients and carriers well known to pharmaceutical chemists.

Design and generation of other therapeutic products

According to the present invention and based on the activity of the antibodies that are produced and characterized herein with respect to IGF-I / II, the design of other therapeutic modalities is facilitated and disclosed to a person skilled in the art. Such modalities include, without limitation, advanced antibody therapeutic products, such as bispecific antibodies, immunotoxins, radiolabeled therapeutic products and V domains of single antibodies, antibody-like binding agent based on frameworks other than region V, generation of peptide therapeutic products. , gene therapies, particularly intrabodies, antisense therapeutic products and small molecules.

In relation to the generation of advanced antibody therapeutic products, in which complement fixation is a desirable attribute, it may be possible to bypass complement dependence to destroy cells through the use of bispecific, immunotoxin or radiolabel agents, for example.

For example, bispecific antibodies comprising (i) two antibodies can be generated, one with a specificity against IGF-I / II and the other against a second molecule, which are conjugated to each other, (ii) a single antibody having a chain specific against IGF-I / II and a second specific chain against a second molecule, or (iii) a single chain antibody that has specificity against both IGF-I / II and the other molecule. Such bispecific antibodies can be generated using techniques that are well known; for example, in relation to (i) and (ii) see for example, Fanger et al. Immunol Methods 4: 72-81 (1994) and Wright and Harris, cited above, and in relation to (iii) see for example, Traunecker et al. Int. J. Cancer (Suppl.) 7: 51-52 (1992). In each case, the second specificity can be prepared as desired. For example, the second specificity can be prepared in heavy chain activation receptors, including, without limitation, CD16 or CD64 (see for example, Deo et al. 18: 127 (1997)) or CD89 (see for example, Valerius et al. Blood 90: 4485-4492 (1997)).

Antibodies can also be modified to act as immunotoxins using techniques that are well known in the art. See for example, Vitetta Immunol Today 14: 252 (1993). See also U.S. Patent No. 5,194,594. In relation to the preparation of radiolabeled antibodies, such modified antibodies can also be easily prepared using techniques that are well known in the art. See for example, Junghans et al. in Cancer Chemotherapy and Biotherapy 655-686 (2nd edition, Chafner and Longo, eds., Lippincott Raven (1996)). See also U.S. Patent Nos. 4,681,581, 4,735,210, 5,101,827,

5,102,990 (RE 35,500), 5,648,471 and 5,697,902. It would be likely that each of the radiolabeled immunotoxins and molecules destroyed cells expressing the oligomerization domain of multimeric enzyme subunits. In some embodiments, a pharmaceutical composition is provided comprising an effective amount of the antibody in association with a pharmaceutically acceptable carrier or diluent.

In some embodiments, an anti-IGF-I / II antibody is bound to an agent (eg, radioisotope, pharmaceutical composition or a toxin). Preferably, such antibodies may be used for the treatment of diseases, such diseases may refer to cells that express IGF-I / II or cells that overexpress IGF-VII. For example, it is contemplated that the drug has the pharmaceutical property selected from the group of antimitotic, alkylating, antimetabolite, antiangiogenic, apoptotic, alkaloid, COX-2 and antibiotic agents and combinations thereof. The drug can be selected from the group of nitrogen mustards, ethyleneimine derivatives, alkylsulfonates, nitrosoureas, triazenes, folic acid analogs, anthracyclines, taxanes, COX-2 inhibitors, pyrimidine analogs, purine analogs, antimetabolites, antibiotics, enzymes, epipodophyllotoxins, platinum coordination complexes, vinca alkaloids, substituted ureas, methylhydrazine derivatives, adrenal suppressors, antagonists, endostatin, taxoles, campotecins, oxaliplatin, doxorubicins and their analogues, and a combination thereof.

Examples of toxins also include gelonin, Pseudomonas exotoxin (PE), PE40, PE38, diphtheria toxin, ricin, ricin, abrin, alpha toxin, saporin, ribonuclease (RNAase), DNase I, staphylococcal enterotoxin A, phytolac antiviral protein americana, gelonin, Pseudomonas endotoxin, as well as derivatives, combinations and modifications thereof.

Examples of radioisotopes include gamma emitters, positron emitters and x-ray emitters that can be used for localization and / or therapy, and beta emitters and alpha emitters that can be used for therapy. The radioisotopes described above as useful for diagnosis, prognosis and stage determination are also useful for therapeutic products. Non-limiting agents of anticancer and antileukemia agents include anthracyclines such as doxorubicin (adriamycin), daunorubicin (daunomycin), idarubicin, detorubicin, carminomycin, epirubicin, esorubicin and morpholino derivatives and substituted, combinations and modifications thereof. Exemplary pharmaceutical agents include cisplatin, taxol, calicheamycin, vincristine, cytarabine (Ara-C), cyclophosphamide, prednisone, daunorubicin, idarubicin, fludarabine, chlorambucil, alpha interferon, hydroxyurea, temozolomide, thalidomide and bleomycin, and combinations and modifications thereof. Preferably, the anticancer or antileukemia is doxorubicin, morpholinodoxorubicin or morpholinodaunorubicin.

As one skilled in the art will appreciate, in the previous embodiments, although affinity values may be important, other factors may be as important or more, depending on the particular function of the antibody. For example, for an immunotoxin (toxin associated with an antibody), the act of binding the antibody to the target may be useful; however, in some embodiments, internalization of the toxin in the cell is the desired end result. As such, antibodies with a high percentage of internalization may be desirable in these situations. Therefore, in one embodiment, antibodies with a high internalization efficiency are contemplated. A high internalization efficiency can be measured as a percentage of internalized antibody, and can be from a low value to 100%. For example, in variable embodiments, a 0.1-5, a 5-10, a 1020, a 20-30, a 30-40, a 40-45, a 45-50, a 50-60, a 60- 70, 70-80, 80-90, 90-99 and 99-100% can be a high efficiency. As one skilled in the art will appreciate, the desirable efficacy may be different in different embodiments, depending on, for example, the associated agent, the amount of antibody that can be administered to a zone, the side effects of the antibody-agent complex, the type (for example, type of cancer) and the severity of the problem to be treated.

In other embodiments, the antibodies disclosed herein provide an assay kit for the detection of IGF-I / II expression in mammalian cells or tissues in order to examine a disease or disorder associated with changes in the IGFI / II expression. The kit comprises an antibody that binds to IGF-I / II and means to indicate the reaction of the antibody with the antigen, if present.

In some embodiments, an article of manufacture is provided comprising a container, comprising a composition containing an anti-IGF-I / II antibody, and a leaflet or label indicating that the composition can be used to treat a disease mediated by IGF-I / II expression. Preferably a mammal, and more preferably a human being, receives the anti-IGFI / II antibody.

Combinations

The anti-IGF-I / II antibodies defined hereinbefore may be applied as a single therapy or may involve, in addition to the compound of the invention, conventional surgery or radiotherapy or chemotherapy. Such chemotherapy may include one or more of the following categories of antitumor agents:

(i)

antiproliferative / antineoplastic drugs and combinations thereof, as used in medical oncology, such as alkylating agents (for example cis-platinum, carboplatin, cyclophosphamide, nitrogen mustard, melphalan, chlorambucil, busulfan and nitrosoureas); antimetabolites (for example antifolates such as fluoropyrimidines like 5-fluorouracil and tegafur, raltitrexed, methotrexate, cytosine arabinoside and hydroxyurea; antitumour antibiotics (for example anthracyclines like adriamycin, bleomycin, doxorubicin, daunomycin, epirubicin, idarubicin, mitomycin-C, dactinomycin and mitramycin); antimitotic agents (for example vinca alkaloids such as vincristine, vinblastine, vindesine and vinorelbine and taxoids such as taxol and taxotere); and topoisomerase inhibitors (for example epipodophyllotoxins such as etoposide and teniposide, amsacrine, topotecan) and camptotecan;

(ii)

 cytostatic agents such as antiestrogens (for example tamoxifen, toremifene, raloxifene, droloxifene and iodoxifene), regulators for decreased estrogen receptor (for example fulvestrant), antiandrogens (for example bicalutamide, flutamide, nilutamide and cyproterone acetate LH) or LHRH agonists (for example goserelin, leuprorelin and buserelin), progestogens (for example megestrol acetate), aromatase inhibitors (for example as anastrozole, letrozole, vorazol and exemestane) and 5a-reductase inhibitors such as finasteride;

(iii) agents that inhibit the invasion of cancer cells (for example metalloproteinase inhibitors such as marimastat and inhibitors of the urokinase plasminogen activator receptor function);

(iv)

 inhibitors of the growth factor function, for example such inhibitors include antibodies against growth factors, antibodies against growth factor receptors (for example, the antierbb2 antibody trastuzumab antibody [HerceptinT] and the anti-erbb1 antibody cetuximab [C225] ), farnesyl transferase inhibitors, MEK inhibitors, tyrosine kinase inhibitors and serine / threonine kinase inhibitors, for example inhibitors of the epidermal growth factor family (eg EGFR family tyrosine kinase inhibitors such as N- (3-Chloro-4-fluorophenyl) -7-methoxy-6- (3-morpholinopropoxy) quinazolin-4-amine (gefitinib, AZD1839), N- (3-ethynylphenyl) -6,7-bis (2-methoxyethoxy) quinazolin-4-amine (erlotinib, OSI-774) and 6-acrylamido-N (3-chloro-4fluorophenyl) -7- (3-morpholinopropoxy) quinazolin-4-amine (CI 1033)), for example family inhibitors of platelet-derived growth factor and for example family inhibitors ia of hepatocyte growth factor;

(v)

antiangiogenic agents such as those that inhibit the effects of vascular endothelial growth factor (for example the anti-vascular endothelial cell growth factor antibody bevacizumab [AvastinT], compounds such as those disclosed in international patent applications WO 97 / 22596, WO 97/30035, WO 97/32856 and WO 98/13354) and compounds that work by other mechanisms (for example linomide, inhibitors of the function of integrin av13, angiostatin and inhibitors of the action of angiopoietin, for example angiopoietin 1 and angiopoietin 2);

(saw)

 vascular damage agents such as combretastatin A4 and compounds disclosed in international patent applications WO 99/02166, WO00 / 40529, WO 00/41669, WO01 / 92224, WO02 / 04434 and WO02 / 08213

(vii) antisense therapies, for example those that target the targets listed above, such as ISIS 2503, an anti-ras antisense;

(viii) gene therapy approaches, including for example approaches to replace aberrant genes such as aberrant p53 or aberrant BRCA1 or GDEPT approaches (gene-directed enzyme prodrug therapy) such as those using cytosine deaminase, thymidine kinase or a bacterial nitroreductase enzyme and approaches that increase the patient's tolerance to chemotherapy or radiotherapy such as gene therapy of multi-drug resistance;

(ix)

 immunotherapy approaches, including for example ex vivo and in vivo approaches to increase the immunogenicity of the patient's tumor cells, such as transfection with cytokines such as interleukin 2, interleukin 4 or macrophage colony stimulating factor, approaches to decrease cell anergy T, approaches that use transfected immune cells such as dendritic cells transfected with cytokines, approaches that use tumor cell lines transfected with cytokines and approaches that use anti-diapathic antibodies;

(x)

 cell cycle inhibitors including for example CDK inhibitors (for example flavopyridol) and other inhibitors of cell cycle control points (for example control point kinase); Aurora kinase and other kinase inhibitors involved in the regulation of cytokinesis and mitosis (eg mitotic kinesins); and histone deacetylase inhibitors;

(xi)

 endothelin antagonists, including endothelin A antagonists, endothelin B antagonists and endothelin A and B antagonists; for example ZD4054 and ZD1611 (WO 96 40681), atrasentán and YM598; Y

(xii) biotherapeutic therapeutic approaches, for example those that use peptides or proteins (such as antibodies or soluble external receptor domain constructs) that either sequester receptor ligands, block ligand binding to the receptor or decrease receptor signaling ( for example due to enhanced degradation of the receptor or reduced expression levels).

Such joint treatment can be achieved by means of simultaneous, sequential or separate dosing of the individual components of the treatment. Such combination products employ the compounds of this invention within the dosage range described hereinbefore and the other pharmaceutically active agent within its approved dosage range.

Examples

The following examples, including the experiments performed and the results achieved are provided for illustrative purposes only and should not be construed to limit the teachings herein.

EXAMPLE 1

Immunization and TITULATION

Immunization

Recombinant human IGF-I and IGF-II antigens obtained from R&D Systems, Inc. (Minneapolis, MN Cat. No. 291-G1 and 292-G2 respectively) were used as antigens. Monoclonal antibodies against IGF-I / II were developed by sequentially immunizing XenoMouse® mice (strains of XenoMouse XMG2 and XMG4 (strain 3C-1), Abgenix, Inc. Fremont, CA). The XenoMouse animals were immunized via the plantar pad for all injections. The total volume of each injection was 50 µl per mouse, 25 µl per plantar pad. A total of ten (10) mice were immunized in each group. Each injection was with 10 [mu] g per mouse of IGF-I or IGF-II alone or conjugated with Californian limpet hemocyanin antigen (KLH) as carrier, as detailed in Table 2. The first PBS injection of Dulbecco (DPBS) and mixed 1: 1 v / v with Titermax Gold adjuvant (SIGMA Cat. No. T2684, Lot No. K1599). A total of 8 to 11 additional reinforcements were then administered over a period of 27 to 38 days, mixed with 25 µg of Adju-Phos (aluminum phosphate gel, catalog No. 1452-250, n. Batch No. 8937, HCl Biosector) and 10 µg of CpG (15 µl of ImmunEasy Mouse adjuvant, catalog no. 303101; lot no. 11553042; Qiagen) per mouse, followed by a final boost of 10! g of pyrogen-free DPBS antigen, without adjuvant. For combined immunization (animals immunized with both IGF-I and IGF-II), the second antigen was administered in the last two (2) boosters.

TABLE 2. SUMMARY OF IMMUNIZATION

Immunization group

Initial immunogen Final immunogen Conjugated with KLH Mice isotype Fusion group

one

IGF-I  IGF-1 - IgG2-KA

one

3

IGF-1 IGF-1 - IgG4-KA one

5

IGF-1 IGF-1 + IgG2-KA one

7

IGF-1 IGF-1 + IgG4-KA one

2

IGF-2 IGF-2 - IgG2-KA

2

4

IGF-2 IGF-2 - IgG4-KA 2

6

IGF-2 IGF-2 + IgG2-KA 2

8

IGF-2 IGF-2 + IgG4-KA 2

9

IGF-1 IGF-2 - IgG2-KA 3

eleven

IGF-1 IGF-2 - IgG4-KA 3

13

IGF-1 IGF-2 + IgG2-KA 3

fifteen

IGF-1 IGF-2 + IgG4-KA 3

10

IGF-2 IGF-1 - IgG2-KA 4

12

IGF-2 IGF-1 - IgG4-KA 4

14

IGF-2 IGF-1 + IgG2-KA 4

16

IGF-2 IGF-1 + IgG4-KA 4

5 EXAMPLE 2

RECOVERY OF LYMPHOCYTES, INSULATIONS OF B-CELLS, FUSIONS AND GENERATION OF HYBRIDOMES

10 Immunized mice were sacrificed by cervical dislocation, and draining lymph nodes were removed and pooled from each cohort. The lymphoid cells were dissociated by milling in DMEM to release the cells from the tissues and the cells were suspended in DMEM. The cells were counted, and 0.9 ml of DMEM per 100 million lymphocytes were added to the cell pellet to carefully resuspend the cells although completely. Using 100 µl of CD90 + magnetic beads per 100 million cells,

15 labeled the cells by incubating the cells with the magnetic beads at 4 ° C for 15 minutes. The magnetically charged cell suspension containing up to 108 positive cells (or up to 2x109 total cells) was loaded on an LS + column and the column was washed with DMEM. The total effluent was collected as the negative fraction for CD90 (most of these cells were expected to be B cells.

The fusion was performed by mixing enriched B cells washed from the above and non-secreting myeloma P3X63Ag8.653 cells acquired from ATCC, cat. CRL 1580 (Kearney et al, J. Immunol. 123, 1979, 15481550) at a ratio of 1: 1. The cell mixture was carefully sedimented by centrifugation at 800 x g. After complete removal of the supernatant, the cells were treated with 2-4 ml of Pronase solution (CalBiochem, cat. No. 53702; 0.5 mg / ml in PBS) for no more than 2 minutes. Then, 3-5 ml of FBS was added to stop the

Enzymatic activity and the suspension was adjusted to 40 ml total volume using cell electrofusion solution, (ECFS, 0.3 M sucrose, Sigma, Cat. No. S7903, 0.1 mM magnesium acetate, Sigma, n Cat. M2545, 0.1 mM calcium acetate, Sigma, Cat. No. C4705). The supernatant was removed after centrifugation and the cells were resuspended in 40 ml of ECFS. This washing step was repeated and the cells were resuspended again in ECFS to a concentration of 2x106 cells / ml.

30 Cell electrofusion was performed using a fusion generator (model ECM2001, Genetronic, Inc., San Diego, CA). The size of the fusion chamber used was 2.0 ml, using the following instrument parameters:

Alignment condition: voltage: 50 V, time: 50 s.

35 Membrane rupture a: voltage: 3000 V, time: 30! S

Retention time after fusion: 3 s.

Following ECF, cell suspensions were carefully removed from the fusion chamber under conditions

5 sterile and transferred to a sterile tube containing the same volume of hybridoma culture medium (DMEM, JRH Biosciences), 15% FBS (Hyclone), supplemented with L-glutamine, pen./estrep., OPI (oxaloacetate , pyruvate, bovine insulin) (all from Sigma) and IL-6 (Boehringer Mannheim). The cells were incubated for 15-30 minutes at 37 ° C, and then centrifuged at 400 x g (1000 rpm) for five minutes. The cells were carefully resuspended in a small volume of hybridoma selection medium (hybridoma culture medium

10 supplemented with 0.5x HA (Sigma, Cat. No. A9666)), and the volume was adjusted appropriately with more hybridoma selection medium, based on a final plate seeding of 5x106 total B cells per 96 plate wells and 200 µl per well. The cells were mixed carefully and pipetted into 96-well plates and allowed to grow. On day 7 or 10, half of the medium was removed, and the cells were fed again with hybridoma selection medium.

15 EXAMPLE 3

SELECTION OF CANDIDATE ANTIBODIES BY ELISA

After 14 days of culture, hybridoma supernatants were examined for specific monoclonal antibodies of IGF-I / II. ELISA plates (Fisher, cat. No. 12-565-136) were coated with 50 µl / well of IGF-I

or human IGF-II (2 µg / ml) in coating buffer (0.1 M carbonate buffer, pH 9.6, NaHCO3 8.4 g / l), then incubated at 4 ° C overnight. After incubation, the plates were washed with wash buffer (0.05% Tween 20 in PBS) 3 times. 200 µl / well of blocking buffer (0.5% BSA, 0.1% Tween 20, was added,

0.01% thimerosal in 1x PBS) and the plates were incubated at room temperature for 1 hour. After incubation, the plates were washed with wash buffer three times. 50 µl / well of hybridoma supernatants, and positive and negative controls were added, and plates were incubated at room temperature for 2 hours.

After incubation, the plates were washed three times with wash buffer. 100 ml / well of

30 goat anti-huIgGFc-HRP detection antibody (Caltag, cat. No. H10507), and the plates were incubated at room temperature for 1 hour. In a secondary examination, the positives were examined in a first examination in two sets, one for the detection of human IgG (heavy chain) and the other for the detection of the human Ig kappa light chain (anti-hIg kappa-HRP of goat (Southern Biotechnology, cat. no. 2060-05) in order to demonstrate the completely human composition for both IgG and Ig kappa After incubation, the

35 plates three times with wash buffer. 100 µl / well of TMB (BioFX Lab. Cat. No. TMSK-010001) was added and the plates were allowed to develop for approximately 10 minutes (until the negative control wells barely began to show color). Then 50 µl / well of stop solution (TMB stop solution, (BioFX Lab. Cat. No. STPR-0100-01) was added and the plates were read in an ELISA plate reader at 450 nm As indicated in Table 3, there were a total of 1,233 wells containing

40 antibodies against IGF-I and II.

All antibodies that bound in the ELISA were counter-examined to detect insulin binding by ELISA in order to exclude those that cross-reacted with insulin. ELISA plates (Fisher, cat. No. 12-565-136) were coated with 50 µl / well of recombinant insulin (concentration: 1 µg / ml;

45 Sigma, catalog No. I2643) in coating buffer (0.1 M carbonate buffer, pH 9.6, NaHCO3 8.4 g / l), then incubated at 4 ° C overnight. As detailed in Table 3, a total of 1,122 antibodies from the original 1233 antibodies cross-reacted with insulin.

TABLE 3. SUMMARY OF EXAM 50

Fn #

Mouse strain Immunogenic Diana with hG detection Diana with hK + hL detection IGF-I (+) and IGF-II (+) IGF-I (+) and IGF-II (+) and human insulin (-)

one

G2 KL IGF-I-KLH IGF-II IGF-I 36 28

2

G4 KL IGF-I-KLH IGF-II IGF-I 65 55

3

G2 KL IGF-II or IGF-II-KLH IGF-I  IGF-II 168 150

4

G4 KL IGF-II or IGF-II-KLH IGF-I  IGF-II 197 194

5

G2KL IGF-I / -II-KLH IGF-II IGF-II 54 fifty

6

G4 KL IGF-I / -II-KLH IGF-II IGF-II 101 86

7

G2 KL IGF-II / -I-KLH IGF-II IGF-II 294 271

8

G4 KL IGF-II / -I or IGF-II / -I-KLH IGF-II  IGF-II 318 288

F1 to F4 total

466 427

F5 to F8 total

767 695

Total

1,233 1,122

Finally, the antibodies that were selected in the counter-examination were then tested by ELISA to confirm binding to mouse IGF-I and IGF-II protein. A total of 683 hybridoma lines were identified that have cross reactivity with mouse IGF-I / II. Therefore, these hybridoma lines expressed

5 antibodies that bound human IGF-I, human IGF-II, mouse IGF-I and mouse IGF-II, but did not bind human insulin.

EXAMPLE 4

10 INHIBITION OF THE IGF-I AND IGF-II UNION TO IGF-IR

The purpose of this study was to examine the 683 anti-IGF-I / II human IgG2 and IgG4 antibodies in the hybridoma supernatant phase to detect neutralizing activity, as determined by inhibition of IGF-I and IGF binding -II to the IGF-IR receptor. Therefore, a receptor / ligand binding assay was performed with cells

NIH3T3 that overexpress the human IGF-IR receptor, as described below.

In summary, Multiscreen filter plates (0.65mM MultiScreen, Millipore 96-well PVDF, Cat. No. MADV N0B 10) were blocked with blocking buffer (PBS containing 10% BSA with 0.02% NaN3 ) at 200 µl / well overnight at 4 ° C. [12I] labeled IGF (Cat. No. of Amersham Life Sciences IM172 (IGF-I) or 20 IM238 (IGF-II)) was diluted to 100 µCi / ml and 50 nM to the appropriate concentration (70 pM final for IGF-I and 200 pM final for IGF-II) in binding buffer (PBS containing 2% BSA with 0.02% NaN3). The filter plate coated with blocking buffer was washed once with 200 µl of PBS, and 50 µl of anti-IGF-I / II Ac supernatants (diluted in binding buffer to a final volume of 25%) were pre-incubated. with 25 µl of [125I] -IGF on the MultiScreen board for 30-60 minutes on ice. Subconfluent NIH3T3 mouse fibroblasts were expressed with trypsin expressing

Stably hIGF-IR (obtained from AstraZeneca) and resuspended in cold binding buffer at 6x106 / ml, and 25 µl of cells were added to the plate for a two-hour incubation on ice. The plate was washed four times with 200 µl of cold PBS and dried overnight. Twenty-five ml / scintillator well (SuperMix cocktail, Cat. No. of Wallac / Perkin Elmer 1200-439) was added and the plates were read using a Trilux Microbeta (Wallac) reader.

30 The following controls were used per test plate: no antibody (total bound IGF), AcM anti-IGF-I (No. 05-172, Upstate) or anti-IGF-II (No. MAB292, R&D Systems) control neutralizers at 50 µg / ml (non-specific background) and 0.075 to 0.5 µg / ml (approximate EC50 values of neutralizing antibodies), and IgM2 mAb (PK16.3.1, Abgenix, no. lot 360-154) or IgG4 (108.2.1, Abgenix, lot # 718-53A) of isotype matching at a concentration of 0.5 µg / ml (approximate EC50 value of neutralizing antibodies). An additional degree of

35 neutralizing control antibodies and human isotype control antibodies matching a plaque per test assay (serial dilution 1/10 from 50 µg / ml (333.3 nM)). All controls were prepared with or without antibodies in binding buffer supplemented with depleted supernatant of anti-KLH human IgG2 or IgG4 at a final volume of 25%.

40 The percent inhibition was determined as follows:

% inhibition = ([Total average CPM of 125I-IGF bound) - (Total average CPM of 125I-IGF bound in the presence of antibody)] / [(Total average CPM of 125I-IGF bound) - (Total average CPM of 125I -IGF bound in the presence of an excess of neutralizing antibody control *)] x 100

* Non-specific background was determined as CPM of cells with an excess of neutralizing anti-IGF Ac control (50 µg / ml, 333.3 nM), which was found to be equivalent to an excess of cold IGF (lower or equal at 10% of total CPM).

50 The anti-IGF-I / II supernatant test was separated by isotype due to radiolabelled ligand availability problems. As shown in Table 4, supernatants of anti-IGF-I / II antibodies with an isotype of IgG2 (total 293) against radiolabeled IGF-I were first examined. A cut-off point at 40% inhibition was initially applied to this test (that is, hybridoma lines were selected that inhibited 40% and above), and 111 hits were selected for subsequent examination against IGF-II. Of the 111 hits, a total of 91 lines were found to inhibit the binding of IGF-II to its receptor with a 50% cut-off point. A total of 71 final hits were selected taking the supernatants that neutralized 50% of the activity of both IGF-I and IGF-II.

5 All supernatants expressing IgG4 (390 total) isotypes against radiolabeled IGF-II were initially examined, and 232 hits were subsequently examined with a 50% cut-off point against IGF-

I. A total of 90 lines could inhibit the binding of IGF-I to its receptor with a 50% cut-off point. After combining the hits for IgG2 (71) and IgG4 (90), a total of 161 lines were obtained that inhibited IGF-I and IGF-II by 50% or more.

10 In conclusion, from the original 683 supernatants, 343 (111 of IgG2 and 232 of IgG4, 50.2%) were selected from the first examination with either IGF-I or IGF-II. A total of 161 final hits (23.6% of the original lines) were obtained, which can block the binding of both IGF-I and IGF-II to IGF-IR with global cut-off criteria of 50% inhibition .

TABLE 4. SUMMARY OF ANTI-IGF-I / II SOLVED OVERRIDE EXAMINATION (50% CUTTING POINT)

ESN activity

Fusion No.

Mouse strain Immunogenic hIGF-II with hG * / hK + or hG + / hL + IGF-I R / L binding assay % of IGF-I + total IGF-II R / L binding assay in IGF-I + % of IGF-I / II + total IGF-II R / L binding assay % of IGF-II + total IGF-I R / L binding assay in IGF-II + % of IGF-I / II + total

one

G2 KL IGF-I-KLH 14 12 13 85.7 26.5 1 4 7.1 8.2 4 86 20.0 75.4 4, 36 20.0 31.6

2

G4 KL IGF-I-KLH twenty

3

G2 KL IGF-II or IGF-II-KLH 49

4

G4 KL IGF-II or IGF-II-KLH 114

5

G2 KL IGF-I / -II-KLH 32 11 75 34.4 37.9 7 59 21.9 29.8 21 121 44.7 57.9 8 42 17.0 20.1

6

G4 KL IGF-V-II-KLH 47

7

G2 KLH IGF-III-I-KLH 198

8

G4 KL IGF-II / -I or IGF-II / -I-KLH 209

683

111 40% cut-off point 16.3 71 10.4 232 34.0 90 13.2

IgG2

IgG4

EXAMPLE 5

ELISAS WITH HIGH CONCENTRATION OF ANTIGEN AND LIMITED CONCENTRATION OF ANTIGEN

5 In order to determine the relative affinities between the 161 hybridoma lines selected in Example 4, as well as the antibody concentration in the supernatants of each line, ELISA assays of high antigen concentration (HA) and concentration were carried out. limited antigen (LA). In the HA quantification assay, high antigen concentration and overnight incubation limit the affinity effect of the antibody, allowing quantification of the relative amount of antigen specific antibody present in each sample.

10 The low concentration of antigen in the LA assay limits the effect of antibody concentration and results in an antibody classification based on its relative affinity.

Quantification test with high antigen concentration

ELISA plates were coated with relatively large amounts of antigen either IGF-I or IGF-II (R&D Systems, Inc., Minneapolis, MN Cat. No. 291-G1 and 292-G2, respectively) at 500 ng / ml (67 nM). Hybridoma supernatants containing antibody were titrated in a dilution range of 1:50 to 1: 12200. A control of a known specific IGF antibody (R&D Systems, Inc., Minneapolis, MN Cat. No. MAB291 and MAB292, respectively) was used to define the linear range of the assay. Then the data was used within the

20 linear range to deduce the relative concentration of the antibody specific for IGF in each titrated sample.

Limited Antigen Concentration Assay

Microtiter plates were coated with low concentrations of antigen. They were added to each well

25 fifty microliters (50 µl) of IGF-I or IGF-II at 64, 32, 16, 8, 4 and 2 ng / ml (covering a range of 8.5 nM to 0.26 nM) in 1% of skim milk / 1X PBS pH 7.4 / 0.5% azide. The plate was incubated for 30 minutes.

The plates were washed four times (4X) with water and 50 µl of hybridoma supernatant containing test antibodies diluted 1.25 in 1% skim milk / 1X PBS pH 7.4 / 0.5 was added to each well % azide Be

30 wrapped the plates tightly with plastic paper or paraffin film, and incubated overnight with stirring at room temperature.

The next day, all plates were washed five times (5X) and 50 µl of goat anti-Fc polyclonal antibody from human IgG-HRP was added to each well at a concentration of 0.5 ug / ml in 1% of milk, 1X PBS pH 7.4.

The plates were incubated for 1 hour at room temperature.

The plates were washed at least five times (5X with tap water). Fifty microliters (50 µl) of HPR-TMB substrate was added to each well and the plate was incubated for 30 minutes. The HRP-TMB reaction was stopped by adding 50 µl of 1 M phosphoric acid to each well. The optical density (absorbance) was measured at 450 nm to

40 each well of the plate.

Data analysis

An average of the OD values of the test antibodies was performed and the interval was calculated. They were selected

45 antibodies with the highest signal and acceptability and low standard deviation as antibodies that had a higher affinity for the antigen than a reference antibody.

An analysis was then performed to select the main antibodies based either on neutralization (example 4), on potency (low antibody concentration as determined by ELISA HA and high 50 ligand binding inhibition), on affinity ( ELISA LA) or in all three criteria. From this analysis, a list of 25 antibodies was generated. A separate analysis based on the% average inhibition of IGF-I and -II binding and affinity for both IGF-I and IGF-II generated a second list of 25 antibodies. Sixteen antibodies were common to both lists, resulting in a final list of 40 antibodies. The results of LA and HA for these 40 antibodies are summarized in Table 5. These 40 lines were selected for cloning, from which

55 successfully cloned 33.

TABLE 5. ELISA RESULTS WITH HIGH CONCENTRATION AND LIMITED ANTIGEN CONCENTRATION FOR 40 MAIN ANTIBODIES

IGF1 HA

IGF2 HA IGF1 LA IGF2 LA IGF1 LA IGF2 LA

ID of

Average Des. Its T. Average Des. Its T. IGF1 IGF2 IGF1 IGF2

line

(ug / ml) HA1 HA1 (ug / ml) HA2 HA2 2 ng / ml 2 ng / ml 4 ng / ml 4 ng / ml

4,121

1.16 0.16 3.56 1.34 0.56 0.53 1.14 0.90

4,141

1.99 0.22 1.99 0.21 0.57 0.79 1.25 1.52

4,142

4.70 0.21 3.65 0.31 0.78 1.00 1.76 1.78

4,143

1.74 0.17 2.03 0.41 0.60 0.99 1.20 1.91

4.25

1.26 0.23 1.48 0.36 0.71 0.81 1.31 1.54

4.69

7.17 1.16 6.50 0.53 0.80 0.80 1.50 1.54

4.90

1.15 0.12 3.68 0.77 0.58 0.48 1.04 0.89

7,118

10.34 1.26 10.32 1.90 0.81 0.85 1.56 1.44

7,123

13.4 3.2 12.0 2.8 1.0 1.7 1.66 2.58

7,127

7.28 0.44 6.59 1.55 1.19 1.37 2.29 2.40

7,130

4.32 0.32 3.51 1.34 0.64 1.17 1.36 1.98

7,146

12.04 0.98 9.63 0.6 0.2 0.86 0.29 1.58

7,158

9.29 0.49 7.1 0.56 1.71 1.42 3.00 2.46

7,159

16.53 1.83 41.1 0.56 1.65 0.98 2.47

7,160

4.9 0.5 5.1 0.2 1.7 2.2 3.14 3.30

7,175

8.46 0.49 6.21 1.22 0.13 0.34 0.14 0.62

7,202

11.94 1.98 15.24 1.72 1.11 1.68 2.28 2.69

7,212

11.30 1.90 10.86 1.26 0.97 0.93 2.22 1.54

7,215

10.11 2.05 10.94 1.39 1.01 1.09 2.25 1.93

7.23

4.30 0.26 3.99 0.29 0.55 1.22 1.40 2.17

7,234

4.7 1.4 3.1 0.4 0.7 1.4 1.79 2.44

7,251

3 0.41 1.93 0.17 1.09 1.02 1.31 1.53

7,252

8.25 0.51 5.55 1.68 1.22 1.23 2.53 2.09

7,268

7.58 0.42 5.07 0.92 1.47 1.37 3.06 2.42

7.29

12.5 1.24 23.53 4.7 0.18 0.39 0.27 0.49

7.3

13.18 2.12 8.83 0.58 0.81 1.28 2.07 1.96

7.34

12.54 1.99 14.67 3.05 0.21 1.07 0.44 1.84

7.41

3.69 0.19 4.97 0.8 1.02 1.53 2.21 2.32

7.56

14.6 2.0 21.7 3.7 1.3 1.4 2.38 2.46

7.58

17.52 0.01 27.54 6.22 0.21 1.15 0.47 1.82

7.66

6.02 0.81 6.18 0.71 0.49 0.97 1.42 1.53

7.77

8.42 0.18 7.25 1.12 0.64 0.46 1.50 1.00

7.85

22.67 0.68 23.63 0.93 0.1 0.33 0.16 0.51

7.99

7.9 0.2 5.9 1.6 0.9 1.0 1.87 1.65

8,119

1.26 0.00 0.77 0.16 2.37 0.79 3.77 1.36

8,141

5.96 0.50 4.12 0.61 1.80 0.61 3.02 1.08

8,146

4.03 0.45 2.55 0.74 0.97 1.06 2.13 1.98

8,287

4.8 0.1 2.8 0.8 2.2 1.6 3.80 2.91

8.8

2.00 0.17 1.45 0.25 1.72 0.77 3.13 1.46

8.86

3.15 0.19 2.36 0.24 0.92 1.35 1.76 2.18

EXAMPLE 6

ANTIBODY UNION TO IGF-1 AND IGF-II UNITED TO IGFBP-3

IGF-I and -II circulate in serum primarily bound to IGF-binding proteins (IGFBP). One objective was to identify antibodies that do not recognize IGF in complex with IGFBP, in order to avoid in vivo depletion of anti-IGF antibodies. The following assay format was developed for the characterization of antibodies that recognize IGF-I or IGF-II when these growth factors complex with IGFBP-3. Specifically, this trial submitted

10 tests the ability of IGF in IGF / anti-IGF antibody complexes to bind IGFBP-3.

Antibody-mediated blocking of IGF capture by IGFBP-3

An assay was developed in which complexes between IGF-I or IGF-II and antibodies specific for IGF were preformed

15 from the examples mentioned above. The ability of these complexes to bind IGFBP-3 was tested using AlphaScreen assay technology (PerkinElmer). In a 384-well plate, 10 µl of hybridoma supernatants diluted 1:20 were mixed with 10 µL of 3 nM biotinylated IGF-I or IGF-II and incubated at room temperature for 2 hours. AlphaScreen donor beads coated with streptavidin and AlphaScreen acceptor beads coupled to IGFPB-3 (10 µL of a mixture, for final dilution were added

20 1/60 of the hybridoma supernatants), and incubation was continued for another hour. The samples were then read on a Packard Fusion plate reader.

Three commercially available anti-IGF monoclonal antibodies M23 (Cell Sciences 05-172 (Upstate) and MAB291 (R&D Systems) showed different capabilities to inhibit IGF binding to IGFBP with IC50 values ranging from a few ng / ml to 100 ng / ml No inhibition of the binding of IGF-I to IGFBP-3 with irrelevant mouse IgG and human IgG up to 10 µg / ml was observed, suggesting that the anti-IGF-I effect is specific. 05-166 (Upstate) and MAB292 (commercially available R&D) showed a significant difference

in affinity for the inhibition of IGF-II / IGFBP-3 interactions. These experiments show that anti-IGF mAbs can block the binding of IGF to IGFBP-3, giving an assay that could be used to examine purified antibodies from hybridoma lines. The next stage was to evaluate the effects of the spent hybridoma medium on the test signal.

5 Serial dilutions of the hybridoma medium and depleted supernatants of anti-KLH hybridoma were tested in the assay system. When the hybridoma supernatants were diluted 1:10 in the preparation for preincubation with IGFI / II (the final dilution in the assay was 1:60), there was almost no effect of the medium on the test results. Based on these data, hybridoma supernatants were diluted for preincubation with IGF, providing the final dilution of 1/60 preferred in the assay.

Six hundred eighty-three depleted supernatants positive for IGF-I and IGF-II binding were examined to determine their ability to inhibit IGF binding to IGFBP-3. Inhibition above 50% for IGF-I and above 60% for IGF-II was used as cut-off criteria. The results summary of the exam using

15 these cut-off points are shown in table 6.

TABLE 6. NUMBERS OF POSITIVE RESULTS IDENTIFIED IN THE EXAM

GF-I

IGF-II IGF-I / II

Samples

Inhibition -> > 50% > 60%

376

(plates 1-4) 48 51 19

307

(plates 5-8) 39 78 32

683

Total 87 129 51

The IGFBP proficiency test using an AlphaScreen assay identified 87 samples that inhibited the binding of IGF-I to IGFBP-3 and 129 samples that inhibited the binding of IGF-II to IGFBP-3 among 683 tested. Fifty-one samples demonstrated double competition from IGF-I and IGF-II. However, in order to more carefully reproduce the function or behavior of the antibodies in vivo, where the IGF and IGFBP complex would be largely preformed, additional tests were performed, as described in example 8.

25 EXAMPLE 7

DETERMINATION OF ANTI-IGF-I AND IGF-II ANTIBODY AFFINANCE USING THE BIACORE ANALYSIS (LOW RESOLUTION EXAMINATION)

Low resolution test of 34 purified monoclonal antibodies

Surface plasmon resonance (SPR) without a marker, or Biacore, was used to measure the affinity of the antibody for the antigen. For this purpose, a high density anti-human goat antibody surface was prepared on a Biacore CM5 chip using a routine amine coupling. All mAbs were diluted until

About 20 µg / ml in HBS-P transfer buffer containing 100 µg / ml BSA. Each AcM was captured on a separate surface using a contact time of 30 seconds at 10 µl / min., And a 5 minute wash for stabilization of the initial AcM level.

IGF-I at 335.3 nM was injected over all surfaces at 23 ° C for 120 seconds, followed by a 5 minute dissociation, using a flow rate of 100 µl / min. Samples were prepared in the HBS-P transfer buffer described above. The surfaces were regenerated after each capture / injection cycle with a 15 second pulse of 146 mM phosphoric acid (pH 1.5). The same capture / injection cycles were repeated for each antibody with 114.7 nM IGF-II. Drift-corrected binding data for the 34 mAbs were prepared by subtracting the signal from a control flow cell and subtracting the initial level drift from a just injected buffer

45 before each antigen injection. The data was set globally to a 1: 1 interaction model using CLAMP to determine binding kinetics (David G. Myszka and Thomas Morton (1998) “CLAMP ©: a binensor kinetic data analysis program,” TIBS 23, 149-150 ). A mass transport coefficient was used in the data adjustment. The results of the kinetic analysis of the binding of IGF-I and IGF-II at 25 ° C are listed in Table 7 below. AcMs are classified from highest to lowest affinity.

TABLE 7. LOW-RESOLUTION BIACORE EXAMINATION OF IGF-I AND IGF-II OF 34 MONOCLONAL ANTIBODIES

IGF-II

IGF-I

Sample

ka (M-1s -1) kd (s-1) KD (pM) ka (M-1s -1) kd (s-1) KD (pM)

7,159.2

3.5 X 106 1.0 X 10-5 2.9 4.3 X 106 9.3 X 10-4 216.0

8.86.1

6.1 X 106 2.4 X 10-4 39.3 3.4 X 106 1.3 X 10-2 3823.0

4.25.1

9.3 X 106 4.1 X 10-4 44.1 5.1 X 106 6.9 X 10-3 1353.0

7,234.2

6.4 X 106 2.9 X 10-4 45.3 6.7 X 106 2.2 X 10-3 328.0

7,160.2

4.6 X 106 2.5 X 10-4 54.3 5.6 X 106 3.3 X 10-3 589.0

7,146.3

3.2 X 106 1.8 X 10-4 56.2 3.8 X 106 8.7 X 10-3 2289.0

7.34.1

3.0 X 106 1.8 X 10-4 60.0 5.2 X 106 3.2 X 10-3 615.0

7,123.1

4.0 X 106 3.4 X 10-4 85.0 4.3 X 106 9.0 X 10-3 2093.0

7,202.3

1.8 X 106 1.7 X 10-4 94.4 1.2 X 106 5.4 X 10-3 4500.0

4,141.1

4.9 X 106 4.7 X 10-4 95.9 * * *

7,215.2

3: 2 X 106 3.3 X 10-4 103.0 3.6 X 106 2.6 X 10-2 7222.0

8,287.2

2.4 X 106 2.5 X 10-4 104. 7.7 X 105 1.3 X 10-3 1688.0

8,146.2

7.7 X 106 8.0 X 10-4 104.0 2.4 X 106 1.3 X 10-2 5417.0

4,143.2

1.1 X 107 1.2 X 10-3 109.0 6.4 X 106 1.9 X 10-2 2969.0

7,251.3

3.5 X 106 4.3 X 10-4 123.0 4.6 X 106 4.3 X 10-3 935.0

7.99.1

6.9 X 106 9.9 X 10-4 143.0 6.0 X 106 4.8 X 10-3 800.0

4,142.2

5.3 X 106 8.5 X 10-4 160.0 8.5 X 106 1.9 X 10-2 2235.0

7.41.3

3.2 X 106 5.5 X 10-4 172.0 5.2 X 106 2.9 X 10-3 558.0

7.56.3

3.3 X 106 6.0 X 10-4 182.0 4.8 X 106 3.1 X 10-3 646.0

7,127.1

4.1 X 106 7.6 X 10-4 185.0 4.9 X 106 3.5 X 10-3 714.0

8.8.3

4.0 X 106 7.8 X 10-4 195.0 3.1 X 106 8.2 X 10-4 264.0

7,158.2

3.4 X 106 6.7 X 10-4 197.0 4.4 X 106 2.2 X 10-3 500.0

7.23.3

3.3 X 106 6.5 X 10-4 197.0 4.1 X 106 5.8 X 10-3 1415.0

7,252.1

3.2 X 106 6.5 X 10-4 203.0 2.3 X 106 2.1 X 10-3 913.0

7.66.1

3.6 X 106 7.7 X 10-4 214.0 2.0 X 106 2.5 X 10-3 1250.0

7.130.1

4.2 X 106 1.0 X 10-3 238.0 4.6 X 106 1.8 X 10-2 3913.0

4.90.2

4.9 X 106 1.2 X 10-3 245.0 * * *

7.3.3

3.2 X 106 8.8 X 10-4 275.0 4.1 X 106 3.9 X 10-3 951.0

7,118.1

4.6 X 106 1.5 X 10-3 326.0 5.6 X 106 5.1 X 10-3 911.0

7.212.1

4.2 X 106 1.6 X 10-3 381.0 3.1 X 106 6.0 X 10-3 1935.0

7.175.2

1.3 X 106 7.4 X 10-4 569.0 1.8 X 106 2.0 X 10-2 11111.0

4,121.1

5.5 X 106 * 3.2 X 10-3 * 582.0 * 1.5 X 106 2.1 X 10-3 1400.0

7.85.2

1.9 X 106 1.3 X 10-3 684.0 2.2 X 106 2.9 X 10-2 13182.0

7.58.3

8.9 X 106 7.9 X 10-3 888.0 7.2 X 106 3.2 X 10-2 4444.0

IGF-I binding data

Most MMAs fit a reasonably well 1: 1 model. The AcM 4,90,2 and

5 4,141.1 through extremely complex data. These mAbs were listed with an asterisk in Table 7 because no significant kinetic constant could be estimated from the model fit. The last phase of dissociation seems to be very slow for both mAbs (at least 1 X 10-5 s-1), which could make these two mAbs useful as therapeutic compounds.

10 IGF-II binding data

Most MMAs fit a reasonably well 1: 1 model. The dissociation for AcM 7.159.2 remained constant at 1 X 10-5 s-1 because there was not enough decomposition data to adequately estimate the kd.

15 The low resolution Biacore studies in this example are designed as a semi-quantitative classification approach. In order to acquire more precise information regarding the characteristic kinetic constants and the affinities of the individual MMAs, high-resolution Biacore studies were carried out as described in Example 8.

20 EXAMPLE 8

DETERMINATION OF ANTIBODY ANTI-IGF-I and IGF-II AFFINITY USING THE BIACORE ANALYSIS (HIGH RESOLUTION EXAMINATION)

25 A high resolution Biacore analysis was performed to further measure antibody affinity for the antigen. Each of the 7.159.2, 7.234.2, 7.34.1, 7.251.3 and 7.160.2 antibodies were captured and each of the IGF-I and IGF-II antigens were injected in a range of concentrations. The resulting binding constants are listed in table 8.

TABLE 8. ANTI-IGF ANTIBODY AFFINENCE DETERMINED BY HIGH AND LOW RESOLUTION BIACORE ANALYSIS

AcM

Low resolution KD (pM) High Resolution KD (PM)

IGF-I

IGF-II  IGF-I IGF-II

7,159.2

216.0 2.9 294.0 1.9

7,234.2

328.0 45.3 3760.0 295.0

7.34.1

615.0 60.0 436.0 421.0 164.0 162.0

7,251.3

935.0 123.0 452.0 47.4

7,160.2

589.0 54.3 2800.0 237.0

Thus, embodiments of the invention may include an antibody that will preferably bind IGF-II, but

5 that will have a cross reaction with IGF-I, joining IGF-II with greater affinity than IGF-I. For example, the antibody can bind IGF-II with an affinity 2.5 times greater than IGF-I. In certain embodiments, the antibody can bind to IGF-II with at least 5, at least 10, at least 25, at least 50 or at least 150 times greater affinity than IGF-I.

10 Examination of preformed IGF-I / GFBP-3 complexes

The IGFBP proficiency test described in Example 6 identified 87 samples that inhibited the binding of IGF-I to IGFBP-3 and 129 samples that inhibited the binding of IGF-II to IGFBP-3 among 683 supernatants tested. Fifty-one samples demonstrated double competition from IGF-I and IGF-II. However, in order to reproduce

15 more carefully the function or behavior of antibodies in vivo, where the IGF and IGFBP complex would be largely preformed, the following Biacore assays were performed on selected antibodies.

Six selected antibodies were examined to determine if they bound IGF-I or IGF-II in complex with IGFBP. The six selected mAbs were covalently immobilized (7,159.2, 7,146.3, 7.34.1, 7,251.3, 7.58.3, and the

20 unrelated ABX-MA1) control antibody at high surface capacity (5,400-12,800 UR) on two Biacore CM5 chips using routine amine coupling with a Biacore 2000 instrument. A flow cell was activated on each CM5 chip and blocked. (without immobilized AcM) for use as a control surface.

Next, IGF-I and IGFBP-3 were mixed together in saline solution buffered with Hepes, pH 7.4, P-20 at

25.005%, 100 µg / ml BSA (HBS-P), until a final solution of 193 nM and 454 nM is obtained, respectively. IGF-II and IGFBP-3 were mixed together to obtain a final solution of 192 nM and 455 nM, respectively. Under these conditions, IGF-I and IGF-II were 99.97% complexed by IGFBP-3. The balance was reached within minutes under these conditions. The solutions of IGF-I / IGFBP-3 and IGF-II / IGFBP-3 were flowed through the various AcM surfaces at 40 µl / min. and 23 ° C, for 180 seconds and a follow-up of

30 dissociation for 120 seconds. Then IGF-I and IGF-II were flowed without complexing through each surface at 193 nM and 192 nM, respectively, and IGFBP-3 was flowed through each surface at 454 nM. The surfaces were regenerated with a 20 second pulse of 10 mM glycine, pH 2.0.

The sensograms were processed using the Scrubber program by subtracting the change in mass refraction index

35 and any non-specific analyte binding signal to the target surface of the surface binding signal with immobilized AcM. After subtraction with white correction, the sensorgrams were taken as a reference for the second time by subtracting an average sensor for buffer injections on a specific flow cell. This "double reference" corrected the AcM binding sensograms for any systematic error present in a particular flow cell.

40 IGF-I / IGFBP-3 and IGF-II / IFBP-3 complexed and uncomplexed were fairly weakly bound to bound antibodies, an approximate estimate of non-specific binding interaction of KD> 1 mM for the six mAbs. , including the ABX-MA1 negative control (see table 9). However, with ABX-MA1, IGF-I / II binding was weak and indicated non-specific binding interactions produced with the three analytes. Apparently, the complexes

45 IGF / IGFBP-3 bind slightly more strongly to all these AcMs than IGFBP-3 alone. However, because both IGF-I, IGF-II and IGBP-3 seem to bind non-specific to these mAbs by themselves, when they bind together, this results in a non-specific protein binding complex. ” more sticky ”, which explains the greater signal of union for the complex. The IGF-I / II / IGFBP-3 and IGFBP-3 complexes bound to the control surface significantly, also indicating the non-specificity of these two proteins. But nevertheless,

50 in the following sensograms this background junction is subtracted from the first reference during data processing, as described above.

This experiment suggests that although it had been previously shown that 51 of the samples inhibited the binding of IGF-I / II to IGFBP3 (example 6), antibodies can also bind to the IGF / IGFBP complex in vitro.

TABLE 9. UNION SUMMARY FOR THE UNION OF IGF-I / IGFBP-3 and IGF-II / IGFBP-3 TO SIX AcM.

AcM

IGFI / IGFBP-3 Complex IGFII / IGFBP-3 Complex IGFBP-3 IGF-I (or II)

7,159.2

+ + + +++

7,146.3

+ ++ + +++

7.34.1

+ ++ ++ +++

7,251.3

++ ++ ++ +++

7.58.3

 ++ ++ ++ +++

ABX-MA1

+ + + +

+, light binding in relation to IGF-I or IGF-II to AcM ++, medium binding in relation to IGF-I or IGF-II to AcM +++, strong binding in relation to IGF-I or IGF binding -II to AcM * These classifications do NOT indicate the KD for these interactions

EXAMPLE 9

5 DETERMINATION OF THE AFFINITY OF ANTI-INSULIN ANTIBODY USING BIACORE ANALYSIS (LOW RESOLUTION EXAMINATION)

The cross-reactivity of antibodies by IGF-I / II was further investigated by measuring the affinity of the AcM for human insulin. Antibodies against IGF-I / II were immobilized to Biacore CM5 chips and insulin was injected into

10 solution for the determination of association and dissociation. In this experiment, five mAbs were tested, including 7,234.2, 7.34.1, 7,159.2, 7,160.2, and 7,251.3. Diluted insulin was injected up to 502 nM in the transfer buffer on all capture surfaces.

No insulin binding was observed to any of the mAbs to 502 nM insulin. These results suggest that there is no apparent cross-reactivity of the mAbs against IGF-I / II with insulin.

EXAMPLE 10

CLASSIFICATION OF ANTIBODIES

20 Epitope classification was performed to determine which of the anti-IGF-I / II antibodies would cross-compete with each other, and therefore would be more likely to bind to the same epitope in IGF-I / II. The classification process is described in US Patent Application 20030175760, also described in Jia et al., J. Immunol. Methods, (2004) 288: 91-98, which are incorporated by reference in their entirety. In short, it

25 coupled Luminex beads with anti-hulgG mouse antibody (Pharmingen # 555784) following the protein coupling protocol provided on the Luminex website. Pre-coupled beads were prepared for coupling to primary unknown antibody using the following procedure, protecting the beads from light. Individual tubes were used for each unknown supernatant. The volume of necessary supernatant was calculated using the following formula: (nX2 + 10) x 50 µl (where n = total number of samples). In this essay you

30 used a concentration of 0.1 µg / ml. The mother of pearl solution was vortexed gently and diluted in supernatant to a concentration of 2500 of each pearl in 50 µl per well or 0.5 X 105 pearls / ml.

Samples were incubated on a shaker in the dark at room temperature overnight.

35 The filter plate was pre-moistened by adding 200 µl of wash buffer per well, which was then aspirated. 50 µl of each bead was added to each well of the filter plate. Samples were washed once by adding 100 µl / well of wash buffer and aspirating. The antigen and controls were added to the filter plate at 50 µl / well. The plate was coated, incubated in the dark for 1 hour on a shaker and then the samples were washed 3 times. Then an unknown secondary antibody was added at 50 µl / well. One was used

Concentration of 0.1 µg / ml for the primary antibody. The plate was then incubated in the dark for 2 hours at room temperature on a shaker, and then the samples were washed 3 times. 50 µl / well of biotinylated anti-human mouse IgG (Pharmingen # 55785) diluted to 1: 500 was added, and the samples were incubated in the dark for 1 hour with stirring at room temperature.

45 Samples were washed 3 times. 50 µl / well of streptavidin-PE was added at a 1: 1000 dilution and the samples were incubated in the dark for 15 minutes with stirring at room temperature. After running two wash cycles on the Luminex100, the samples were washed 3 times. The contents of each well were resuspended in 80 µl of blocking buffer. The samples were carefully mixed with pipetting several times to resuspend the beads. The samples were then analyzed in the Luminex100. The results are presented at

50 continued in table 10.

TABLE 10. CLASSIFICATIONS FOR 34 ANTIBODIES AGAINST IGF-I / II MAIN POSITIVES IN FUNCTIONAL TEST

IGF-1

IGF-II Classification one

Sort 2 Sort 3 Unclassified

Sort 1 Sort 2 Sort 3 Unclassified

7.3.3

7.58.3

7.175.2

7,215.2

7.3.3

7,158.2

7.175.2

7,215.2

7.23.3

8,287.2

7.85.2

7,127.1

8,146.2

4.90.2

7,661

4.90.2

7.99.1

7,252.1

4,141.1

7.56.3

4,141.1

7,123.1

8.86.1

7.85.2

7,160.2

7,146.3

7.212.1

7,251.3

7.41.3

7.34.1

7,234.2

7,159.2

4,121.1

7,159.2

7.130.1

7,146.3

8,146.2

7,251.3

7,118.1

7.34.1

7,252.1

8,287.2

7,123.1

7.58.3

7.212.1

7.66.1

7,234.2

7.41.3

7.99.1

7.56.3

7,127.1

7,160.2

4.25.1

7,202.3

8.8.3

8.8.3

7,158.2

4.25.1

7,202.3

7.23.3

7.130.1

4,142.2

8.86.1

4,143.2

4,142.2

4,121.1

7,118.1

4,143.2

EXAMPLE 11

STRUCTURAL ANALYSIS OF ANTI-IGF-I / II ANTIBODIES

5 Variable heavy chains and variable light chains of several antibodies were sequenced to determine their DNA sequences. Full sequence information for anti-IGF-I / II antibodies is provided in the sequence list with nucleotide and amino acid sequences for each combination of gamma and kappa chains. Variable heavy sequences were analyzed to determine the VH family, the

10 sequence of the D region and the sequence of the J region. The sequences were then translated to determine the primary amino acid sequence and compared to the sequences of the VH, D and J regions of the germ line to evaluate somatic hypermutations.

The alignment of the sequences of these antibodies with the germline genes is shown in the tables.

15 following. Table 11 is a table that compares the heavy chain regions of the antibody with the heavy chain region of its related germ line. Table 12 is a table that compares the regions of the kappa light chain of the antibody with the region of the light chain of its related germ line. Mutations that move away from the germ line are shown as the new amino acid.

20 Variable regions (V) of immunoglobulin chains are encoded by multiple germline DNA segments, which bind into functional variable regions (VHDJH or VKJK) during cell ontogeny

B. The molecular and genetic diversity of the antibody response to IGF-I / II was studied in detail. These assays revealed several specific points for anti-IGF-I / II antibodies.

The analysis of five individual antibodies specific for IGF-I / II resulted in the determination that the antibodies were derived from three different germline VH genes, four of them from the VH4 family, resulting in 2 segment antibodies VH4-39 gene. Tables 11 and 12 show the results of this analysis.

30 It should be noted that the amino acid sequences between sister clones collected from each hybridoma are identical. For example, the heavy chain and light chain sequences for AcM 7.159.2 are identical to the sequences shown in Tables 11 and 12 for AcM 7.159.1.

The heavy chain CDR1 of the antibodies of the invention have a sequence as disclosed

35 in table 11. The CDR1 disclosed in table 11 are from the definition of Kabat. Alternatively, CDR1 can be defined using an alternative definition to include the last five residues of the FR1 sequence. For example, for antibody 7.159.1 the FR1 sequence is QVQLVQSGAEVKKPGASVKVSCKAS (SEQ ID NO .: 93) and the CDR1 sequence is GYTFTSYDIN (SEQ ID NO .: 94); for antibody 7.158.1 the sequence of FR1 is QLQLQESGPGLVKPSETLSLTCTVS (SEQ ID NO .: 95) and the sequence of CDR1 is GGSIRSSSYYWG (SEQ ID NO .: 96); for antibody 7.234.1, the FR1 sequence is QLQLQESGPGLVKPSETLSLTCTVS (SEQ ID NO .: 97) and the CDR1 sequence is GGSINSSSNYWG (SEQ ID NO .: 98); for antibody 7.34.1 the sequence of FR1 is QVQLQESGPGLVKPSETLSLTCTVS (SEQ ID NO .: 99) and the sequence of CDR1 is GGSISSYYWS (SEQ ID NO .:

5 100); and for antibody 7.251.3, the FR1 sequence is QVQLQESGPGLVKPSETLSLTCTVS (SEQ ID NO .: 101) and the CDR1 sequence is GGSISSYYWS (SEQ ID NO .: 102).

It should also be appreciated that when a particular antibody differs from the sequence of its respective germ line at the amino acid level, the antibody sequence can be mutated back to the sequence to the germ line. The 10 corrective mutations of this type can occur in one, two, three or more positions, or in a combination of any of the mutated positions, using conventional molecular biology techniques. By way of non-limiting example, Table 12 shows that the sequence of the light chain of AcM 7.34.1 (SEQ ID NO .: 12) differs from the sequence of the corresponding germ line (SEQ ID NO.:80) by one Pro to Ala mutation (mutation 1) in the FR1 region, and by a Phe to Leu mutation (mutation 2) in the FR2 region. Thus, the 15 or nucleotide amino acid sequence encoding AcM 7.34.1 light chain can be modified to change mutation 1 to give the germ line sequence at the site of mutation 1. In addition, the amino acid sequence or nucleotides encoding the light chain of AcM 7.34.1 can be modified to change mutation 2 to give the germline sequence at the site of mutation 2. Still further, the amino acid or nucleotide sequence encoding the light chain of AcM 7.34.1 can be modified to change both mutation 1

20 as mutation 2 to give the germline sequence at the sites of both mutations 1 and 2.

TABLE 11. HEAVY CHAIN ANALYSIS

String name

SEQ ID NO. V D J FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4

Germ line **

75 VH1-8 N.D. JH6B

7_159_1

6 " " v

Germ line

77 VH4-39  D6-19 JH2

7_158_1

2 " " "

7_234_1

18 " " "

Germ line

79 VH4-59  D1-20 JH6B

7_34_1

10 " " "

7_251_3

14 " " "

* The pad designation (#) indicates a space in the germ line and is used to show proper alignment with the antibody sequences shown in the table. ** The germline sequences shown in the table above are for alignment purposes and it should be understood that each individual antibody region exists at its own location within the variable regions of the immunoglobulin germline DNA segments in vivo. .

TABLE 12. LIGHT CHAIN ANALYSIS

String name

SEQ ID NO. V J FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4

Germ line

76 VH1-19 JL2

7_159_1

8 " v

Germ line

78 L5 JK3

7_158_1

4 " "

7_234_1

twenty " "

Germ line

80 V1-13 JL 2

7_34_1

12 " "

7_251_3

16 n "

* The pad designation (#) indicates a space in the germ line and is used to show proper alignment with the antibody sequences shown in the table. ** The germline sequences shown in the table above are for alignment purposes and it should be understood that each individual antibody region exists at its own location within the variable regions of the immunoglobulin germline DNA segments in vivo. .

EXAMPLE 12 INHIBITION OF PHOSPHORILATION INDUCED BY IGF-I AND IGF-II OF HIGF-IR EXPRESSED ECTOPICALLY

IN NIH3T3 CELLS

5 IGF ligands exert their proliferation and anti-apoptosis functions by activating receptor tyrosine kinase activity in the IGF-IR receptor. In order to assess the ability of anti-IGF-I / II antibodies to inhibit IGF-IR induced IGF phosphorylation, NIH3T3 cells that ectopically express hIGF-IR were used in the following assay.

10 NIH3T3 cells that ectopically express human IGF-IR were seeded in a 96-well plate at a density of 10,000 cells per well and incubated overnight in starvation medium (FBS treated with 1% charcoal). The next day, the growth medium was discarded, the wells were gently washed twice with PBS, and 100 µL of serum free medium (0% FBS) was added to starve the cells.

After 1-2 hours, 100 µl of 0.05% BSA-free serum medium containing either IGF-I (10 nM) or IGF-II (10 nM) was added to the cells in triplicate. it had been pre-incubated for 60 minutes at 37 ° C with various concentrations of antibody. Stimulation was allowed to occur for 10 minutes at 37 ° C. After stimulation, the media was removed and 100 ul of 3.7% formaldehyde in PBS / 3% BSA was added to each well and incubated at RT for 20 min. The 2X cells were then washed with PBS and added to

20 wells 100 ul permeabilization buffer (0.1% Triton-X in 3% BSA / PBS). This was allowed to incubate at RT for 10 min., Discarded and 100 ul of 0.6% hydrogen peroxide in PBS / 3% BSA was added to quench any endogenous peroxidase activity. After incubation at RT for 20 min., 3X cells were washed with 0.1% PBS / Tween-20 and blocked by adding 100 ul of 10% FBS in PBS / 0.1% Tween-20 at RT for 1 h. The blocking buffer was then removed and 50 ul of anti-phospho antibody was added to each well

25 IGFIR at 1 ug / ml (Cat. No. 44-804, BioSource) in 10% FBS / PBS-T. After incubation at RT for 2 h, 3X cells were washed with PBST by immersing for 5 minutes between each wash. After washing, 50 ul / well of a secondary goat anti-rabbit IgG-HRP Fc region antibody diluted 1: 250 in blocking buffer was added to each well. After 1 hour of incubation at RT, 3X cells were washed for 5 minutes with PBST as before and dried by tapping them. Then 50 ul of ECL reagent was added

30 (DuoLux) and the URLs were read immediately.

Thirty-two (32) antibody lines were examined and two independent assays were performed for each antigen. The results for the ten main antibodies are summarized in table 13 below.

35 TABLE 13. SUMMARY OF INHIBITION OF IGF-IR FOSFORILATION DEPENDENT ON IGF IN NIH3T3 CELLS

Results of IGF-I pTYR (n = 2)

Results of IGF-II pTYR (n = 2)

Max activation%

EC50 (nM) *  Max activation% EC50 (nM) *

AcM ID

Half FROM Half FROM Half FROM Half FROM

7,159.2

16.5% 6.6% 7.4 0.8 28.6% 5.4% 3.1 0.2

7.34.1

14.2% 1.8% 9.4 0.8 21.5% 3.9% 2.5 0.1

7,146.3

19.5% 3.9% 19.0 5.7 23.6% 2.1% 3.6 0.2

7,251.3

16.9% 5.4% 14.5 0.7 15.9% 1.5% 3.0 0.9

7,234.2

21.1% 3.0% 24.3 1.0 21.1% 0.1% 7.7 0.1

7,160.2

33.6% 6.4% 22.9 0.1 21.5% 0.6% 4.7 0.2

7,158.2

22.7% 0.9% 28.3 0.5 33.7% 2.4% 11.3 3.2

7.56.3

31.3% 2.2% 25.1 4.3 21.2% 0.2% 6.3 0.5

7,118.1

24.1% 5.2% 40.8 2.6 21.8% 3.2% 13.9 5.3

7.41.3

33.1% 6.1% 47.1 7.0 29.5% 4.4% 3.5 4.0

* These tests may have been performed under antigen limiting conditions given the KD of the AcM for IGF-I and IGF-II.

EXAMPLE 13

40 INHIBITION OF THE PROLIFERATION INDUCED BY IGF-I and IGF-II OF NIH3T3 CELLS TRANSFECTED WITH HIGF-IR

As previously mentioned, one of the criteria for neutralizing antibodies against IGF-I / II is the ability to inhibit IGF-induced proliferation. In order to assess the ability of antibodies to determine IGF-induced proliferation, NIH3T3 cells expressing ectopically hIGF-IR were used in the following assay.

NIH3T3 cells that express ectopically hIGF-IR were seeded in a 96-well plate at a density of

5,000 cells per well and were grown overnight in starvation medium (FBS treated with charcoal

one%). The next day, the growth medium was discarded, the wells were gently washed twice in serum-free medium, and 100 µL of serum-free medium was added to starve the cells. 100 µl of starvation medium containing 15 ng / ml of IGFI or 50 ng / ml of IGFII pre-incubated for 30 min were added to the cells in duplicate or triplicate. at 37 ° C with various antibody concentrations. After 20 hours of incubation,

5 pulsed the cells with BdrU for 2 hours and the degree of incorporation (proliferation) was quantified using the Roche cell proliferation ELISA kit (Roche, Cat. No. 1,647,229).

A total of 32 antibody lines were examined and two or three independent assays were performed for each antigen. The results for the ten main antibodies are summarized in table 14 below.

10 TABLE 14. SUMMARY OF IGF DEPENDENT PROLIFERATION INHIBITION IN NIH3T3 / hIGF-IR CELLS

IGF-I Proliferation Assay

IGF-II Proliferation Assay

% inhibition

EC50 (nM) *  % inhibition EC50 (nM) *

AcM ID

Mean (n = 3) FROM Mean (n = 3) FROM Mean (n = 2) FROM Mean (n = 2) FROM

7,159.2

77.0% 9.6% 24.1 5.9 99.3% 0.6% 7.6 2.5

7.34.1

72.6% 5.6% 23.4 8.1 73.6% 11.8% 16.3 0.4

7,146.3

65.3% 5.5% 37.2 4,5 82.0% 6.9% 15.9 3.4

7,251.3

72.4% 15.3% 38.9 4.3 79.2% 7.8% 22.0 3.7

7,234.2

67.3% 6.9% 40.6 4.6 62.1% 17.2% 24.3 2.4

7,160.2

62.8% 5.7% 47.6 10.7 45.9% 0.8% 24.7 2.8

7,158.2

57.4% 19.5% 42.8 1.7 54.6% 6.6% 36.0 4.2

7.56.3

50.2% 7.8% 65.7 31.9 48.0% 10.9% 38.3 7.5

7,118.1

59.4% 14.5% 1626.6 2714.5 68.3% 0.8% 49.9 3.8

7.41.3

29.5% 14.7% 76.3 35.9 51.9% 13.7% 61.9 23.7

* These tests may have been performed under antigen limiting conditions given the KD of the AcM for IGF-I and IGF-II.

EXAMPLE 14 INHIBITION OF PHOSPHORILATION INDUCED BY IGF-I AND IGF-II OF HIGF-IR EXPRESSED IN CELLS OF

BxPC3 HUMAN PANCREATIC TUMOR

IGF-IIII exerts its proliferation and anti-apoptosis functions by activating the receptor tyrosine kinase activity in the

20 IGF-IR receiver. In order to evaluate the ability of the antibodies to inhibit IGF-IR induced IGF phosphorylation, human pancreatic tumor cells BcPC3, which express endogenous hIGF-IR, were used in the following assay.

BxPC3 cells were seeded in a 96-well plate at a density of 55,000 cells per well and were

25 incubated overnight in regular growth medium. The next day, the growth medium was discarded, the wells were gently washed twice in serum-free medium, and 1000 µl of serum-free medium was added to the cells to starve the cells. After 24 hours, the medium was discarded and the cells were gently washed once in medium without serum. Serum free medium was pre-incubated with 0.05% BSA containing either IGF-I (20 ng / ml) or IGF-II (75 ng / ml) for 30 minutes at 37 ° C with various antibody concentrations, and

30 then 100 µl was added to the cells in triplicate. The plates were incubated for 15 minutes at 37 ° C, and subsequently rinsed with cold PBS. 100 µl of lysis buffer was added to the wells and the plates were incubated for 30 minutes at 4 ° C. The lysates were centrifuged at 2000 rpm for 10 minutes at 4 ° C, and the supernatant was collected. Phosphorylation of IGF-IR was quantified using the Duoset human phosphorus-IGF-IR ELISA kit (R&D Systems, Cat. No. DYC1770).

35 Ten antibody lines were examined and two independent assays were performed for each antigen. The results are summarized in table 15 below.

TABLE 15. SUMMARY OF INHIBITION OF IGF-IR FOSFORILATION DEPENDENT ON IGF 40 NIH3T3

Results of IGF-I pTYR (n = 2)

Results of IGF-II pTYR (n = 2)

n = 1

n = 2  n = 1 n = 2

AcM ID

% inhibition max. (333.3 nM) EC50 (nM) % inhibition max. (333.3 nM) EC50 (nM) % inhibition max. (333.3 nM) EC50 (nM) % inhibition max. (133.3 nM) EC50 (nM)

7,159.2

100.0 3.3 100.0 1.6 100.0 1.6 91.2 1.7

7.34.1

100.0 5.9 98.5 3.8 100.0 2.0 89.7 1.9

7,146.3

96.4 16.1 94.2 10.7 100.0 2.0 87.9 2.0

7,251.3

95.7 7.5 95.2 5.3 100.0 N.D. 91.3 2.6

7,234.2

97.3 5.1 91.5 2.9 98.5 1.9 77.3 2.3

7,160.2

93.4 5.3 89.2 3.1 88.6 1.7 73.2 2.5

7,158.2

92.9 4,5 89.4 3.6 92.4 N.D. 74.0 4.2

7.56.3

84.9 N.D. 88.7 6.5 91.4 10.1 66.2 5.1

7,118.1

90.5 13.1 90.6 11.8 95.7 17.9 78.0 13.1

7.41.3

88.6 6.5 86.5 6.5 88.6 4,5 70.6 3.1

* 333 nM for the last 3 antibodies. N.D .: Not determined

EXAMPLE 15 INHIBITION OF PROLIFERATION INDUCED BY IGF-I AND IGF-II OF PANCREATIC TUMOR CELLS

5 HUMAN BxPC3

As previously mentioned, one of the criteria for neutralizing antibodies against IGF is the ability to inhibit IGF-induced proliferation. In order to assess the ability of antibodies to inhibit IGF-induced proliferation, BcPC3 human pancreatic tumor cells, which express hIGF-IR, were used.

10 endogenous, in the following test.

BxPC3 cells were seeded in a 96-well plate at a density of 2,000 cells per well and grown overnight in regular growth medium. The next day, the growth medium was discarded, the wells were gently washed twice in serum free medium, and 100 µl of serum free medium was added with

Transferrin 10 µg / ml and 0.1% BSA (assay medium) to produce starvation of the cells. After 24 hours, the medium was discarded and the cells were gently washed once in serum-free medium, and 100 µl of test medium containing 20 ng / ml IGF pre-incubated for 30 minutes was added to the cells in duplicate or in triplicate at 37 ° C with various antibody concentrations. Plates were incubated for 3 days and proliferation was quantified using the CellTiter-Glo reagent (Promega).

20 Ten antibody lines were examined and two or three independent assays were performed for each antigen. The results are summarized in table 16 below. Based on the functional data below and the data in Example 1.4, the four best antibodies were selected. Data from the IGF-I-induced proliferation test were excluded from the selection criteria due to the high variability of the observed trial.

25 TABLE 16. SUMMARY OF IGF-DEPENDENT PROLIFERATION INHIBITION IN HUMAN PANCREATIC TUMOR CELLS BxPC3

Proliferation results with IGF-II

AcM ID

% inhibition max. (333.3 nM) EC50 (nM) % inhibition max. (133.3 nM) EC50 (nM)

7,159.2

120.0 0.8 118.3 0.9

7.34.1

117.0 0.5 109.0 6.7

7,146.3

128.7 3.0 119.5 7.3

7.2513

128.5 0.5 105.3 4.4

7,234.2

111.7 N.D. 200.7 2.6

7,160.2

79.7 1.1 155.7 N.D.

7,158.2

86.3 0.0013 148.3 N.D.

7.56.3

87.0 N.D. 112.3 102.0

7,118.1

114.0 34.0 137.0 54.7

7.41.3

102.0 N.D. 73.0 N.D.

* 333 nM for the last 3 antibodies. N.D .: Not determined

EXAMPLE 16 30 DETERMINATION OF REACTIVITY CROSSED WITH IGF-1, IGF-II AND MOUSE INSULIN

One objective was to develop antibodies that were specific for IGF-I and IGF-II but did not have cross-reactivity with insulin. In order to perform subsequent experiments on animals, the antibodies must also cross-react with murine IGFI / II but not with murine insulin. Therefore, ELISA assays were performed to determine if the selected antibodies could cross-react with IGF

or murine insulin.

As shown in Table 17, five of the ten major antibodies were tested for cross-reactivity with mouse or rat insulin by ELISA. The ELISAs showed that these antibodies did not have cross-reactivity with mouse or rat insulin, compared to the negative control antibody PK16,3.1 and compared to the positive anti-rat insulin control antibody.

TABLE 17. CROSS REACTIVITY WITH MOUSE INSULIN

DO at 450 with different Ag

Antibodies

Mouse insulin Rat insulin Without Ag

7,159.2

0.52 0.52 0.56

7,160.2

0.60 0.57 0.62

7.34.1

0.48 0.47 0.55

7,251.3

0.55 0.53 0.56

7,234.2

0.51 0.49 0.66

Serum

1.28 1.23 1.34

Rat anti-insulin

2.52 3.06 0.10

PK16.3.1

0.58 0.58 0.62

EXAMPLE 17 10 INHIBITION OF PHOSPHORILATION INDUCED BY IGF-I AND IGF-II MOUSE OF HUMAN IGF-IR

EXPRESSED ECTOPICALLY IN NIH3T3 CELLS

Monoclonal antibodies with cross-reactivity with mouse IGF-I and IGF-II were further tested in order to determine the extent to which they inhibit IGF-IR IGF-induced phosphorylation. This essay is

15 performed as described above using NIH3T3 cells that ectopically express the hIGF-IR receptor. The results of this test are summarized in table 18.

NIH3T3 cells that ectopically express human IGF-IR were seeded in a 96-well plate at a density of 10,000 cells per well and incubated overnight in starvation medium (FBS treated with 1% charcoal). The next day, the growth medium was discarded, the wells were gently washed twice with PBS, and 100 µL of serum free medium (0% FBS) was added to starve the cells. After 1-2 hours, 100 µl of 0.05% BSA-free serum medium containing either IGF-I (10 nM) or mouse IGF-II (10 nM) was added to the cells in triplicate (R&D Systems, Inc., Minneapolis, MN Cat. No. 791-MG and 792-MG, respectively) which had been pre-incubated for 60 minutes at 37 ° C with various concentrations of antibody. Stimulation was allowed to occur for 10 minutes at 37 ° C. After stimulation, the media was removed and 100 ul of 3.7% formaldehyde in PBS / 3% BSA was added to each well and incubated at RT for 20 min. The 2X cells were then washed with PBS and 100 ul of permeabilization buffer (0.1% Triton-X in 3% BSA / PBS) was added to each well. This was allowed to incubate at RT for 10 min., Discarded and 100 ul of 0.6% hydrogen peroxide in PBS / 3% BSA was added to quench any endogenous peroxidase activity. After incubation at RT for 20 min., 3X cells were washed with 0.1% PBS / Tween-20 and blocked by adding 100 ul of 10% FBS in PBS / 0.1% Tween-20 at RT for 1

h. The blocking buffer was then removed and 50 ul of 1 µg / ml anti-phospho-IGFIR antibody (cat. No. 44-804, BioSource) in 10% FBS / PBS-T was added to each well. After incubation at RT for 2 h, 3X cells were washed with PBST by immersing for 5 minutes between each wash. After washing, they were added to each

35 of the 50 ul / well wells of a goat anti-Fc region secondary antibody of rabbit IgG-HRP diluted 1: 250 in blocking buffer. After 1 hour of incubation at RT, 3X cells were washed for 5 minutes with PBST as before and dried by tapping them. Then 50 ul of ECL reagent (DuoLux) was added and the URLs were read immediately.

40 TABLE 18. INHIBITION OF PHOSPHORILATION INDUCED BY HIGF-IR MOUSE IGF

Mouse IGF-I EC50 (nM)

Mouse IGF-II EC50 (nM)

AcM ID

n = 1 n = 2 n = 1 n = 2

7,159.2

2.8 5.7 3.1 5.0

734.1

6.0 10.2 4.0 9.7

7,251.3

6.7 10.6 5.4 8.7

7,234.2

46.0 36.1

7,160.2

49.5 225.2

EXAMPLE 18

INHIBITION OF THE GROWTH OF NIH3T3 CELLS THAT EXPRESS IGF-II AND IGF-1R LIVE IN MOUSES 45 NUDES

In order to assess the ability of antibodies to inhibit IGF-II induced proliferation in vivo, the following experiments were performed.

6-8 weeks old female nude mice (provided by Charles River Laboratories, Wilmington, MA, USA) were implanted subcutaneously with 5 x 106 cells of clone 32 (NIH3T3 cells that ectopically overexpress human IGF-II). Human IGF-1R). The cells were suspended in PBS in a total inoculum volume of 330 µl. Tumors were allowed to grow to 100-200 mm 3 before treatment with monoclonal antibodies 7.159.2, 7.34.1 and 7.251.3. The antibodies or the IgG2 isotype control antibody suspended in PBS was administered intraperitoneally to randomized groups of 9 or 12 mice weekly for 4 weeks at 5 or 50 mg / kg from day 22. PBS was administered as a vehicle control to a Additional group of 11 mice weekly for 4 weeks from day 22. Tumor size and body weight were measured 2-3 times per week. The results are summarized in Figures 1 and 2.

Significant inhibition of tumor growth was observed (see Figure 1) without significant weight loss in any group (see Figure 2). The 7,159.2, 7.34.1 and 7.251.3 antibodies significantly inhibited the growth of clone tumors 32 to 5 and 50 mg / kg / week (see Figure 1).

EXAMPLE 19

INHIBITION OF THE GROWTH OF NIH3T3 CELLS THAT EXPRESS IGF-I and IGF-1R IN VIVO IN NAKED MOUSES

In the previous example, the antibodies were shown to inhibit IGF-II induced proliferation in vivo. In order to evaluate the ability of antibodies to inhibit IGF-I induced proliferation in vivo, the following experiments were performed.

5 x 106 viable P12 cells [NIH3T3 cells that ectopically overexpress human IGF-I and human IGF-1R (Pietrzkowski et al. , Cell Growth & Differentiation, 3, 199-205, 1992)] subcutaneously on the left side. The cells were suspended in PBS in a total inoculum volume of 0.1 ml. Two groups of animals (each of n = 10) were administered twice a week from the day of implantation of the NIH3T3 cells or the AcM 7.159.2 at 1.0 mg per mouse or an equivalent vehicle volume PBS (0.3 ml) with the same program. All doses were administered intraperitoneally through the route (i.p.). The body weights of the animals were measured daily, and once established, tumor measurements were taken twice a week using calipers. The volume of all measurable tumors was calculated from the calibrator measurements assuming an ovoid shape. The results are summarized in Figure 3.

As shown in Figure 3, significant inhibition of tumor growth was observed with AcM 7.159.2 after i.p. twice a week of 1.0 mg of antibody / mouse. No significant weight loss was observed in any of the animal groups.

EXAMPLE 20

INHIBITION OF GROWTH OF TUMOR CELLS IN HUMAN PATIENTS

A group of human cancer patients who were diagnosed with pancreatic cancer in treatment groups is randomized. Each group of patients is treated with weekly intravenous injections of AcM 7.159.2, 7.34.1 or

7,251.3 described herein. An effective amount of the antibody ranging from 50 mg / kg to 2,250 mg / kg is administered to each patient for 4-8 weeks. Only a conventional chemotherapeutic regimen is administered to a control group.

At periodic times during and after the treatment regimen, the tumor burden is evaluated by magnetic resonance imaging (MRI). It is found that patients who have received weekly antibody treatment with AcM 7.159.2, 7.34.1 or 7.251.3 show significant reductions in tumor size, compared to patients who do not receive antibody treatment. In some treated patients, the tumors are no longer detectable. In contrast, tumor size increases or remains substantially the same in the control group.

EXAMPLE 21

INHIBITION OF GROWTH OF TUMOR CELLS IN A HUMAN PATIENT

A human patient with a malignant tumor is diagnosed. The patient is treated with weekly intravenous injections of AcM 7.159.2 for 8 weeks. At periodic times during and after the treatment regimen, the tumor burden is evaluated by magnetic resonance imaging (MRI). Significant reductions in tumor size are found.

EXAMPLE 22

TREATMENT OF ACROMEGALIA IN A HUMAN PATIENT An adult man of acromegaly is diagnosed. The patient is treated with intravenous injections twice a day.

week of AcM 7.34.1 over a period of 2 years. As a result, the patient experiences a significant reduction in acromegaly symptoms. EXAMPLE 23 TREATMENT OF PSORIASIS IN A HUMAN PATIENT An adult woman is diagnosed with severe psoriasis. The patient is treated with intravenous injections twice

per week of AcM 7,251.3 over a period of 3 weeks. As a result, the patient experiences a significant reduction in psoriasis symptoms. EXAMPLE 24

TREATMENT OF OSTEOPOROSIS IN A HUMAN PATIENT An adult woman is diagnosed with osteoporosis. The patient is treated with intravenous injections twice a week of AcM 7.159.2 for a period of one year. As a result, there is a significant reduction in bone density loss.

EXAMPLE 25 TREATMENT OF ATEROSCLEROSIS IN A HUMAN PATIENT An adult man is diagnosed with atherosclerosis. The patient is treated with intravenous injections twice a

the week of AcM 7.34.1 over a period of one year. As a result, the patient experiences a reduction in atherosclerosis symptoms, such as angina pectoris. EXAMPLE 26

TREATMENT OF RESISTANCE IN A HUMAN PATIENT An adult man undergoes angioplasty to relieve a blocked artery. Following the angioplasty procedure, the patient is treated with intravenous injections twice a week of AcM 7,251.3 for a period of one year. As a result, the patient does not experience restenosis of the treated artery.

EXAMPLE 27 TREATMENT OF DIABETES IN A HUMAN PATIENT An adult woman is diagnosed with diabetes. The patient is treated with intravenous injections twice a day.

week of AcM 7.159.2 over a period of one year. As a result, the symptoms of diabetes are reduced. SEQUENCES Subcloned hybridomas were sequenced to determine their primary structure both at the nucleotide level

as of amino acids for both the heavy chain variable genes and for the light chain variable genes. The nucleotide and polypeptide sequences of the variable regions of the monoclonal antibodies against IGF-I and IGF-II, as listed in Table 1, are given in the sequence list.

EQUIVALENTS The above written specification is considered sufficient to allow a person skilled in the art to practice the invention. The description and examples above detail certain preferred embodiments of the invention and describe the best mode contemplated by the inventors. It will be appreciated, however, that regardless of how detailed the foregoing may appear in the text, the invention can be practiced in many ways and the invention must be interpreted according to the appended claims.

Sequence list

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Claims (18)

1. Fully human isolated specific binding protein that preferentially binds to insulin-II growth factor (IGF-II) with cross-reactivity for insulin-like growth factor I (IGF-I) and neutralizes IGF activity -I and IGF-II, said binding protein, or binding fragment thereof: a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of "Ser Tyr Asp Ile Asn" (SEQ ID NO: 33); a heavy chain complementarity determining region 2 (CDR2) having the amino acid sequence of "Trp Met Asn Pro Asn Ser Gly Asn Thr Gly Tyr Ala Gln Lys Phe Gln Gly" (SEQ ID NO: 34); a heavy chain complementarity determining region 3 (CDR3) having the amino acid sequence of "Asp Pro Tyr Tyr Tyr Tyr Tyr Gly Met Asp Val" (SEQ ID NO: 35); a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of "Ser Gly Ser Ser Ser Asn Ile Glu Asn Asn His Val Ser" (SEQ ID NO: 36); a light chain complementarity determining region 2 (CDR2) having the amino acid sequence of "Asp Asn Asn Lys Arg Pro Ser" (SEQ ID NO: 37); Y a light chain complementarity determining region 3 (CDR3) having the amino acid sequence of "Glu Thr Trp Asp Thr Ser Leu Ser Ala Gly Arg Val" (SEQ ID NO: 38).

2.

Specific binding protein according to claim 1, said binding protein being monoclonal antibody 7.159.2 (ATCC registration number PTA-7424).

3.

Specific binding protein according to claim 1, said binding protein comprising a heavy chain polypeptide having the sequence of SEQ ID NO .: 6;

or said binding protein comprising a light chain polypeptide having the sequence of SEQ ID NO .: 8.

Four.

Specific binding protein according to any one of claims 1 or 3, said binding protein comprising a heavy chain polypeptide having the sequence of SEQ ID NO .: 6;

and said binding protein comprising a light chain polypeptide having the sequence of SEQ ID NO .: 8.

5.

Specific binding protein according to any one of the preceding claims, said binding protein being a fully human monoclonal antibody; or

a binding fragment of a fully human monoclonal antibody.

6.

Specific binding protein according to claim 5, wherein said binding fragment is selected from the group consisting of Fab, Fab ’or F (ab’) 2 and Fv.

7.

Specific binding protein according to any one of claims 1 to 6, in a mixture with a pharmaceutically acceptable carrier.

8.

Nucleic acid molecule encoding specific binding protein according to any one of claims 1 to 6.

9.

Vector comprising the nucleic acid molecule according to claim 8.

10.

Host cell comprising the vector according to claim 9.

eleven.

Specific binding protein according to any one of claims 1 to 6, said specific binding protein not specifically binding to IGF-II or IGF-I proteins when said proteins are bound to insulin growth factor binding proteins.

12.

Method for determining the level of insulin-II growth factor (IGF-II) and insulin-like growth factor I (IGF-I) in a sample of a patient comprising:

contacting a sample of a patient with the specific binding protein according to any one of claims 1 to 6; Y determine the level of IGF-I and IGF-II in said sample.

13.

Use of the specific binding protein according to any one of claims 1 to 6 in the preparation of a medicament for the treatment of a malignant tumor.

14.

Use according to claim 13, wherein said malignant tumor is selected from the group consisting of: melanoma, non-small cell lung cancer, glioma, hepatocellular carcinoma (liver), thyroid tumor, gastric (stomach) cancer, cancer of prostate, breast cancer, ovarian cancer, bladder cancer, lung cancer, glioblastoma, endometrial cancer, kidney cancer, colon cancer, pancreatic cancer and squamous cell carcinoma.

fifteen.

Use according to claim 13 or 14, wherein said medicament is for use in combination with a second antineoplastic agent selected from the group consisting of an antibody, a chemotherapeutic agent and a radioactive drug.

16.

Use according to claim 13 or 14, wherein said medicament is for use in conjunction with or after conventional surgery, a bone marrow stem cell transplant or a peripheral stem cell transplant.

17.

Conjugate comprising the antibody according to claim 5 or a binding fragment thereof and a therapeutic agent.

18.

Conjugate according to claim 17, wherein the therapeutic agent is a toxin;

a radioisotope; or A pharmaceutical composition